HUMAN iNKT CELL ACTIVATION USING GLYCOLIPIDS WITH ALTERED GLYCOSYL GROUPS

ABSTRACT

Glycosphingolipids (GSLs) bearing α-glucose (α-Glc) that preferentially stimulate human invariant NKT (iNKT) cells are provided. GSLs with α-glucose (α-Glc) that exhibit stronger induction in humans (but weaker in mice) of cytokines and chemokines and expansion and/or activation of immune cells than those with α-galactose (α-Gal) are disclosed. GSLs bearing α-glucose (α-Glc) and derivatives of α-Glc with F at the 4 and/or 6 positions are provided. Methods for iNKT-independent induction of chemokines by the GSL with α-Glc and derivatives thereof are disclosed. Methods for immune stimulation in humans using GSLs with α-Glc and derivatives thereof are provided.

RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.14/480,521, filed on Sep. 8, 2014, which claims priority to U.S.Provisional Patent Application No. 61/874,946, entitled HUMAN iNKT CELLACTIVATION USING GLYCOLIPIDS WITH ALTERED GLYCOSYL GROUPS, filed on Sep.6, 2013, the contents of which are hereby incorporated by reference asif set forth in their entirety.

FIELD OF THE INVENTION

This invention relates generally to the field of immune therapeutics. Inparticular, the instant disclosure relates to glycolipids and variantsthereof that modulate invariant natural killer T (iNKT) cells in humansand stimulate cytokine and/or chemokine production and thustransactivate downstream immune cells thereby bridging the innate andadaptive immunity.

BACKGROUND OF THE INVENTION

Natural killer-like T (NKT) cells are a distinct population of Tlymphocytes with enormous therapeutic potential in the treatment ofdiseases such as cancer and autoimmune disorders. Invariant naturalkiller T (iNKT) cells form a subset of regulatory T cells with featuresof both innate and adaptive immunity. In contrast to conventional Tcells that are activated by a peptide presented by an MHC class I or IImolecule, iNKT cells recognize lipid derivatives present in the contextof CD1d, a non-classical MHC I molecule expressed on antigen presentingcells (APCs).

α-GalCer (α-galactosylceramide), a glycolipid composed of a-linkedgalactose, sphingosine, and an acyl tail, was obtained by structuraloptimization of agelasphin, which is isolated from the marine spongeAgelas mauritianus. It has been found to exhibit antitumor activity bothin vitro and in vivo and shown to be the most potent ligand yet knownfor both mouse and human invariant natural killer T cells (iNKT cells).

Invariant NKT cells (iNKT cells) carry the invariant TCR-α chain(Vα14/Jα18 in mice and Vα24/Jα18 in humans) and co-express CD161 antigen(NK cell marker NK1.1 in mice and NKR-P1A in humans). (1) Lantz, O.;Bendelac, A. J. Exp. Med. 1994, 180, 1097; (2) Dellabona, P.; Padovan,E.; Casorati, G.; Brockhaus, M.; Lanzavecchia, A. J. Exp. Med. 1994,180, 1171; (3) Makino, Y.; Kanno, R.; Ito, T.; Higashino, K.; Taniguchi,M. Int. Immunol. 1995, 7, 1157; and (4) Davodeau, F.; Peyrat, M. A.;Necker, A.; Dominici, R.; Blanchard, F.; Leget, C.; Gaschet, J.; Costa,P.; Jacques, Y.; Godard, A.; Vie, H.; Poggi, A.; Romagne, F.;Bonneville, M. J. Immunol. 1997, 158, 5603. They secrete large amountsof Th1 (e.g., IFN-γ, IL-2) and Th2 (e.g., IL-4, IL-6) cytokines inresponse to αGalCer presented by the CD1d molecule on theantigen-presenting cells.⁵⁻⁹ (5) Kawano, T.; Cui, J.; Koezuka, Y.;Toura, I.; Kaneko, Y.; Motoki, K.; Ueno, H.; Nakagawa, R.; Sato, H.;Kondo, E.; Koseki, H.; Taniguchi, M. Science 1997, 278, 1626; (6)Yoshimoto, T.; Paul, W. E. J. Exp. Med. 1994, 179, 1285; (7) Arase, H.;Arase, N.; Nakagawa, K.; Good, R. A.; Onoe, K. Eur. J. Immunol. 1993,23, 307; (8) Kawakami, K.; Yamamoto, N.; Kinjo, Y.; Miyagi, K.;Nakasone, C.; Uezu, K.; Kinjo, T.; Nakayama, T.; Taniguchi, M.; Saito,A. Eur. J. Immunol. 2003, 33, 3322; and (9) Nieuwenhuis, E. E.;Matsumoto, T.; Exley, M.; Schleipman, R. A.; Glickman, J.; Bailey, D.T.; Corazza, N.; Colgan, S. P.; Onderdonk, A. B.; Blumberg, R. S. Nat.Med. 2002, 8, 588. These secreted cytokines could then transactivatedownstream immune cells, including dendritic cells (DC), natural killercells (NK), B cells, CD4⁺ T and CD8⁺ T cells, and thereby bridging theinnate and adaptive immunity.¹⁰⁻¹² (10) Eberl, G.; MacDonald, H. R. Eur.J. Immunol. 2000, 30, 985; (11) Eberl, G.; Brawand, P.; MacDonald, H. R.J. Immunol. 2000, 165, 4305; and (12) Kitamura, H.; Ohta, A.; Sekimoto,M.; Sato, M.; Iwakabe, K.; Nakui, M.; Yahata, T.; Meng, H.; Koda, T.;Nishimura, S.; Kawano, T.; Taniguchi, M.; Nishimura, T. Cell. Immunol.2000, 199, 37.

However, the counterbalance of Th1 and Th2 cytokines may limit theclinical application of αGalCer for the treatment of a variety ofdisorders.¹³⁻¹⁶ (13) Tahir, S. M.; Cheng, O.; Shaulov, A.; Koezuka, Y.;Bubley, G. J.; Wilson, S. B.; Balk, S. P.; Exley, M. A. J. Immunol.2001, 167, 4046; (14) Dhodapkar, M. V.; Geller, M. D.; Chang, D. H.;Shimizu, K.; Fujii, S.; Dhodapkar, K. M.; Krasovsky, J. J. Exp. Med.2003, 197, 1667; (15) Giaccone, G.; Punt, C. J.; Ando, Y.; Ruijter, R.;Nishi, N.; Peters, M.; von Blomberg, B. M.; Scheper, R. J.; van derVliet, H. J.; van den Eertwegh, A. J.; Roelvink, M.; Beijnen, J.;Zwierzina, H.; Pinedo, H. M. Clin. Cancer Res. 2002, 8, 3702; and (16)Bricard, G.; Cesson, V.; Devevre, E.; Bouzourene, H.; Barbey, C.; Rufer,N.; Im, J. S.; Alves, P. M.; Martinet, O.; Halkic, N.; Cerottini, J. C.;Romero, P.; Porcelli, S. A.; Macdonald, H. R.; Speiser, D. E. J.Immunol. 2009, 182, 5140.

SUMMARY OF THE INVENTION

Accordingly, many analogues were designed to stimulate selective Th1 orTh2 cytokine responses of iNKT cells. Glycolipids with marked Th1 biasin both mice and men, leading to superior tumour protection in vivo. Forexample, glycosphingolipids (GSLs) with the truncated sphingosine tailcould drive immune responses into Th2 direction and prevented autoimmuneencephalomyelitis.¹⁷ Miyamoto, K.; Miyake, S.; Yamamura, T. Nature 2001,413, 531. On the other hand, GSL with phenyl ring on the acyl chaininduced Th1-biased cytokines in mice and humans and displayed morepotent anticancer activities against breast, lung and melanoma tumors inmice.^(18,19) (18) (Chang, Y. J.; Huang, J. R.; Tsai, Y. C.; Hung, J.T.; Wu, D.; Fujio, M.; Wong, C. H.; Yu, A. L. Proc. Natl. Acad. Sci.U.S.A. 2007, 104, 10299 and (19) Wu, T. N.; Lin, K. H.; Chang, Y. J.;Huang, J. R.; Cheng, J. Y.; Yu, A. L.; Wong, C. H. Proc. Natl. Acad.Sci. U.S.A. 2011, 108, 17275.

Examination of the binary interaction between CD1d and glycolipids, aswell as the ternary interaction between iNKT TCR and CD1d-glycolipidcomplex elucidated the mechanisms underlying their structure-activityrelationships (SAR). As compared to αGalCer, phenyl GSLs with the sameglycosyl group exhibited stronger binary and ternary interactions,leading to more Th1-biased responses, and the biological responses had asignificant correlation with the binding avidities of the ternarycomplex both in mice and humans.19-21 (19) Wu, T. N.; Lin, K. H.; Chang,Y. J.; Huang, J. R.; Cheng, J. Y.; Yu, A. L.; Wong, C. H. Proc. Natl.Acad. Sci. U.S.A. 2011, 108, 17275; (20) Liang, P. H.; Imamura, M.; Li,X.; Wu, D.; Fujio, M.; Guy, R. T.; Wu, B. C.; Tsuji, M.; Wong, C. H. J.Am. Chem. Soc. 2008, 130, 12348; and (21) Li, X.; Fujio, M.; Imamura,M.; Wu, D.; Vasan, S.; Wong, C. H.; Ho, D. D.; Tsuji, M. Proc. Natl.Acad. Sci. U.S.A. 2010, 107, 13010.

Glycosphingolipids (GSLs) bearing α-galactosyl group (αGal) and phenylring on the acyl chain were found to be more potent than α-galactosylceramide (αGalCer) to stimulate invariant NKT (iNKT) cells. Theiractivities in mice and humans correlated well with the binding aviditiesof the ternary interaction between iNKT TCR and CD1d-GSL complex.

Invariant natural killer T (iNKT) cells are known to have markedimmunomodulatory capacity due to their ability to produce copiousamounts of effector cytokines. There is a need for improvedglycosphingolipids that stimulate human invariant NKT (iNKT) cells andmodulate cytokine and chemokine production in humans.

Accordingly, the present disclosure is based on the unexpected discoverythat glycosphingolipids (GSLs) with α-glucose (α-Glc) exhibit strongerinduction in humans (but weaker in mice) of cytokines and chemokines andexpansion and/or activation of immune cells than those with α-galactose(α-Gal). The change of the 4′-OH direction on the glycosyl group led todifferent bioactivities in mice and humans. Alterations at the 6position of the glycosyl group also showed variable effects on thebiological responses. GSLs with α-Glc and derivatives of α-Glc with F atthe 4 and/or 6 positions are disclosed herein. Methods foriNKT-independent induction of chemokines by the GSL with α-Glc andderivatives of α-Glc are disclosed. Methods for immune stimulation inhumans using GSLs with α-Glc and derivatives of α-Glc thereof areprovided.

The present disclosure provides a method for augmenting animmunogenicity of an antigen in a subject in need thereof, comprisingcombined administration said antigen as conadministration orcoformulation with an adjuvant composition comprising a GSLs of thegeneral Formula 1.

According to the present invention, the use of GSLs as an immuneadjuvant results in an enhancement and/or extension of the duration ofthe protective immunity induced by the antigen and is attributed atleast in part to the enhancement and/or extension of antigen specificTh1-type responses.

The GSLs-α-Glc-containing adjuvant of the invention can be conjointlyadministered with any antigen, in particular, with antigens derived frominfectious agents or tumors. Preferably, the adjuvant and antigen areadministered simultaneously, most preferably in a single dosage form.

In a further embodiment, the invention provides a prophylactic and/ortherapeutic method for treating a disease in a subject comprisingadministering to said subject an immunoprotective antigen together withan adjuvant composition that includes GSLs. As specified herein, thismethod can be useful for preventing and/or treating various infectiousor neoplastic diseases.

In conjunction with the methods of the present invention, also providedare pharmaceutical and vaccine compositions comprising animmunogenically effective amount of an antigen and an immunogenicallyeffective amount of an adjuvant selected from GSLs within Formula 1 aswell as, optionally, a pharmaceutically acceptable carrier or excipient.

Accordingly, in one aspect, the present disclosure relates to structuraland functional exemplars of immune adjuvant compounds of Formula (I):

or pharmaceutically acceptable salt thereof; wherein R¹, R², R³, R⁴, R⁵,n and m are as described herein.

In some embodiments, R⁴ is of Formula (II):

wherein i and R⁶ are as described herein.

In some embodiments, R⁴ is of Formula (III):

wherein j, k, R⁷ and R⁸ are as described herein.

Aspects of the disclosure also relates to pharmaceutical compositionscomprising (i) a compound disclosed herein in an amount sufficient tostimulate an immune response in when administered to a subjects,including humans, and (ii) a pharmaceutically acceptable excipient.

In some embodiments, the pharmaceutical composition comprises an antigenand a vaccine adjuvant. In certain embodiments, the antigen is a tumorantigen.

In some embodiments, the pharmaceutical composition comprises ananti-cancer therapeutic.

In some embodiments of the pharmaceutical composition, R⁴ in thecompound is selected from substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl, and wherein the compound iscapable of increasing Th1 cytokines in humans with minimum accompanyingincrease in Th2 cytokines.

Aspects of the invention relates to methods for stimulating an immuneresponse in a human subject in need thereof, the method comprising:administering to the subject a therapeutically effective amount of acomposition disclosed herein.

In some aspects, the compound is administered in amount capable ofelevating invariant Natural Killer T (iNKT) cells in humans.

In some aspects, administration of the compound increases cytokineand/or chemokine production in humans. In some embodiments, the cytokineproduction is sufficient to transactivate downstream immune cells. Insome embodiments, the downstream immune cells comprise one or more ofdendritic cells (DC), natural killer cells (NK), B cells, CD4⁺ T andCD8⁺ T cells.

In some aspects, the cytokines comprise Th1 cytokines. In someembodiments, the Th1 cytokines are selected from: interferon-gamma(IFN-γ), GM-CSF, TNFα, interleukin 2, interleukin 12 and interleukin 10.

In some aspects, the chemokines are selected from: RANTES, MIP-1α, KC,MCP-1, IP-10 and MIG.

In some aspects, administration of the composition has an anti-cancereffect. In some embodiments, the cancer is selected from the groupconsisting of lung cancer, breast cancer, hepatoma, leukemia, solidtumor and carcinoma.

In some embodiments, R⁴ in the compound is selected from substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl, andwherein increase in Th1 cytokines in humans exceeds any increase in Th2cytokines.

Aspects of the invention relates to methods for elevating invariantNatural Killer T (iNKT) cells production in a human subject in needthereof, the method comprising: administering to the subject atherapeutically effective amount of a composition, wherein thecomposition comprises a compound disclosed herein. In some embodiments,the elevation of iNKT levels is greater when compared to the elevationresulting from administration of an equivalent amount of a glycolipidanalogue comprising alpha-galactose (αGal) as the glycosyl head group.

Aspects of the invention relates to methods for stimulating cytokineand/or chemokine production in a human subject in need thereof, themethod comprising: administering to the subject a therapeuticallyeffective amount of a composition, wherein the composition comprises anamount sufficient to increase cytokine/chemokine production, of acompound disclosed herein.

In some aspects, cytokine production is sufficient to transactivatedownstream immune cells. In some embodiments, the downstream immunecells comprise one or more of dendritic cells (DC), natural killer cells(NK), B cells, CD4⁺ T and CD8⁺ T cells.

In some aspects, the cytokines comprise Th1 cytokines. In someembodiments, the cytokines are selected from: interferon-gamma (IFN-γ),GM-CSF, TNFα, interleukin 2, interleukin 12 and interleukin 10.

In some aspects, the chemokines are selected from: RANTES, MIP-1α, KC,MCP-1, IP-10 and MIG.

These and other aspects will become apparent from the followingdescription of the preferred embodiment taken in conjunction with thefollowing drawings, although variations and modifications therein may beaffected without departing from the spirit and scope of the novelconcepts of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, the inventions of which can be better understood byreference to one or more of these drawings in combination with thedetailed description of specific embodiments presented herein.

FIG. 1A shows: The structures of glycolipids with αGal or αGlc.7DW8-5-Man is the only compound with αMan.

FIG. 1B shows: Synthesis of glycolipid analogues, reagents andconditions were as follows. a, Me2S, 2-Cl-pyridine, Tf20, CH2Cl2, 4 &Aring; MS, −45° C., 16 h, 50-60%. b, Ph3P, pyr., H2O, 50° C., 9 h. c,fatty acids, EDC, HBTu, Et3N, CH2Cl2, 60%, two steps. d, AcOH—H₂O 4:1,65° C., 16 h. e, NaOME, MeCH—CH2Cl2 1:4, 5 h. f, Pd(OH)2, H2,MeOH—CH2Cl2 1:1, 5 h, 20-30%, three steps.

FIGS. 2A-D show: Glycolipid analogs with αGal were stronger immunemodulators than those with αGlc in mice but weaker in humans. B6 wildtype and Jα18 knockout mice (n=4) were i.v. injected with the indicatedglycolipid (1 μg/mouse) or vehicle. Sera collected at 2 h and 18 hpost-injection were analyzed for the secretion of cytokines like IFN-γ(2A) and IL-4 (2B). IFN-γ and IL-4 peaked at 18 hr and 2 hrpost-injection, respectively. (2C) B6 WT mice treated with the indicatedglycolipid (1 μg/mouse) or vehicle (1% DMSO in PBS) were sacrificed at72 hr post-injection and their splenocytes were subjected to FACSanalysis for the determination of total CD3+ cells. (2D) Human naïveVα24+ iNKT cells were cultured with autologous immature CD14+ DCs pulsedwith the indicated glycolipid at 100 ng/ml or DMSO on day 2. Afterantigen removal on day 3, human iNKT cells were cultured in the presenceof IL-2. The number of expanded iNKT cells was counted using the GuavaViaCount reagent on day 9. Student t test was chosen to analyze thedifference between 7DW8-5 and 7DW8-5-Glc with p<0.001, ***. Assays wereperformed in two different donors with similar trends.

FIGS. 3A-D show: The ternary interaction of CD1d-glycolipid complex withiNKT cells. (3A) DN3A4-1.2 Vα14+ iNKT hybridoma cells and (3B)7DW8-5-expanded Vα24+ iNKT cells were incubated with variousconcentrations of the indicated dimeric mCD1d-glycolipid andhCD1d-glycolipid complexes for 30 min at 4° C., respectively. The levelof bound complexes at the indicated concentration was detected byanti-mIgG1 secondary antibody and analyzed by flow cytometry. Therelationship between the binding percentage and the concentration ofCD1ddi-glycolipids complex was plotted in mice (3A) and humans (3B). KDvalues in mice (3C) and humans (3D) were calculated from Scatchardtransformation of the plot (3A) and (3B), respectively. Assay wasperformed in duplicates.

FIGS. 4A-B show: mCD1d vs. hCD1d swapping assay. (4A) Murine DN3A4-1.2Vα14+ iNKT hybridoma cells or (4B) C1-expanded Vα24+ iNKT cells werepulsed with the indicated glycolipid presented by either mCD1d (A20-CD1dcells) or hCD1d (HeLa-CD1d cells) at 1, 0.1, and 0.01 μg/ml. After 18hr, the supernatants were harvested to measure IL-2 secretion by anELISA assay (4A) or using Beadlyte® Human Cytokine kit and Luminex® 200™reading system (4B). Assays were performed in triplicates. 8-5 was theabbreviation of 7DW8-5.

FIGS. 5A-E show: Computer modeling of the ternary complex ofCD1d-GSL-iNKT TCR. (5A)/(5B) hydrogen bonds within the CD1d-C1-iNKT TCRcomplex of mice (5A) and humans (5B) were shown. Formation of hydrogenbonds was noted in the conserved residues, including human Asp80 (mouseAsp80), human Thr154 (mouse Thr156), human Asp151 (mouse Asp 153) ofCD1d and human Gly96 (mouse Gly96) of iNKT TCR. Besides, mouse Asn30 aswell as human Phe29 and Ser30 of iNKT TCR were the key residues formingH-bond interactions with 3′- and/or 4′-OHs of C1. (5C) the equatorial4′-OH of glucose could compensate for the loss of Phe29 interaction by astronger interaction with a crystal water, which was trapped by humaniNKT TCR-Phe51 and hCD1d-Trp153. (5D) the higher energy from aromaticinteractions could drive the acyl chain of C34 or C34-Glc to a lowerposition (near Cys12) of the A′ channel within CD1d, leading to a subtleperturbation to the orientation of the head group. (5E) The computedfree energy of the ternary complex using Autodock4.2

FIGS. 6A-D show: Dose-dependent chemokine secretions triggered by7DW8-5-Glc. B6 wild type mice were i.v. injected with 7DW8-5-Glc at 0.1or 1 μg/mouse. Sera collected at 2 h and 18 h post-injection wereanalyzed for chemokine secretions such as IP-10 (6A), KC (6B), MCP-1(6C), and MIP-1α (6D). These chemokines peaked at 2 hr post-injection.

FIGS. 7A-H show: iNKT-dependent productions of cytokines and chemokines.B6 wild type and Jα18 knockout mice were i.v. injected with theindicated glycolipids (1 μg/mouse) or vehicle. Sera collected at 2 h and18 h post-injection were analyzed for cytokines like IL-2 (7A), IL-6(7B), GM-CSF (7C) and TNFα (7D) as well as chemokines such as IP-10(7E), MIG (7F), KC (7G) and MCP-1 (7H). Only MIG peaked at 18 hrpost-injection, while the others peaked at 2 hr post-injection.

FIGS. 8A-F show: FACS analyses of WT mouse immune cells after theindicated glycolipid stimulation. B6 WT mice treated with the indicatedglycolipid (1 μg/mouse) or vehicle (1% DMSO in PBS) were sacrificed at72 hr post-injection and their splenocytes were subjected to FACSanalysis. (8A) The total splenocytes, (8B) total CD11Chi cells, (8C)CD11Chi/CD80+ cells, (8D) CD11Chi/CD86+ cells, (8E) CD4+ T cells and(8F) CD8+ T cells.

FIGS. 9A-F show: FACS analyses of Jα18 KO mouse immune cells after theindicated glycolipid stimulation. B6 Jα18 KO mice treated with theindicated glycolipid (1 μg/mouse) or vehicle (% DMSO in PBS) weresacrificed at 72 hr post-injection and their splenocytes were subjectedto FACS analysis. (9A) The total splenocytes, (9B) total CD11Chi cells,(9C) CD11Chi/CD80+ cells, (9D) CD11Chi/CD86+ cells, (9E) CD4+ T cellsand (9F) CD8+ T cells (student t test: *, p<0.05, as compared to D)

FIGS. 10A-D show: Binding strengths of the binary complex between mCD1dand glycolipid. (10A, 10B) Different concentrations of mCD1d-glycolipidcomplexes coated on the ELISA plate were incubated with the saturatedamount of L363 antibody conjugated with biotin, followed bystreptavidin-HRP detection and ELISA measurement. (10A) The relationshipbetween OD values reflecting L363 antibody binding and the concentrationof CD1ddi-glycolipids complex was plotted. (10B) The dissociationconstant (KD) between L363 antibody and the indicated mCD1d-glycolipidcomplex was calculated as described in Materials and methods. (10C) Therelationship between OD values reflecting L363 antibody binding and theconcentration of glycolipids was plotted. (10D) KD values of the binarycomplex were calculated from the linear regression of the Scatchardtransformation of the L363 antibody binding curve (10C).

FIGS. 11A-D show: CD1d dimer staining of in vivo C1-pulsed splenocytes.B6 WT splenocytes (n=3) were harvested 3 days after injection with C1 (1μg/mouse) and stained with CD3, CD45R and the indicated dimer complexconjugated with RPE for 1 hr at 4 degrees Celsius. (11A) CD3+/CD45R−cells were gated to analyze the dimer staining. (11B) unloaded dimer wasused as the control. (11C) mCD1d dimer loaded with 7DW8-5-Glc stained17.1±0.8% of C1-pulsed splenocytes. (11D) mCD1d dimer loaded with 7DW8-5stained 36.2±5.0% of C1-pulsed splenocytes.

FIGS. 12A-C show: mCD1d vs. hCD1d swapping assay. C1-expanded Vα24+ iNKTcells were pulsed with the indicated glycolipid antigen presented byeither mCD1d (A20-CD1d cells) or hCD1d (HeLa-CD1d cells) at 1, 0.1, and0.01 μg/ml. After 18 hr, the supernatants were harvested for themeasurement of IFN-γ (12A) and IL-4 (12B) secretions. (12C) The ratio ofIFN-γ over IL-4 was calculated at different concentrations ofglycolipids. The ratio of IFN-γ over IL-4 from different glycolipidswere compared to that from C1 at the indicated concentrations by studentt test (*, p<0.05; **, p<0.01; ***, p<0.001). Assays were performed intriplicates.

DETAILED DESCRIPTIONS

Natural killer T cells (NKTs) represent a subset of T lymphocytes withunique properties, including reactivity for natural or syntheticglycolipids presented by CD1d and expression of an invariant T cellantigen receptor (TCR) alpha chain. NKTs are different from functionallydifferentiated conventional αβ T cells in that they share properties ofboth natural killer cells and T cells are can rapidly produce bothTH1-type and TH2-type responses upon stimulation with their ligands(innate immunity). The activation of NKTs paradoxically can lead eitherto suppression or stimulation of immune responses. For example, theproduction of TH1 cytokines is thought to promote cellular immunity withantitumor, antiviral/antibacterial, and adjuvant activities, whereas TH2cytokine production is thought to subdue autoimmune diseases and promoteantibody production. Because NKTs play a regulatory role in the immunesystem, they are attractive targets for immunotherapy.

As stated above, the present disclosure is based on the unexpecteddiscovery that glycosphingolipids (GSLs) with α-glucose (α-Glc) exhibitstronger induction in humans (but weaker in mice) of cytokines andchemokines and expansion and/or activation of immune cells than thosewith α-galactose (α-Gal). Accordingly, methods and compositionscomprising exemplary GSLs are provided herein.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Culture Of Animal Cells (R.I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes(IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning(1984); the treatise, Methods In Enzymology (Academic Press, Inc.,N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P.Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology,Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell AndMolecular Biology (Mayer and Walker, eds., Academic Press, London,1987); Antibodies: A Laboratory Manual, by Harlow and Lanes (Cold SpringHarbor Laboratory Press, 1988); and Handbook Of Experimental Immunology,Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986). In addition,the methods of making and using immune adjuvants are described in U.S.Pat. Nos. 7,488,491 and 7,928,077, the relevant disclosures of which areincorporated by reference herein.

As used herein, the term “lipid” refers to any fat-soluble (lipophilic)molecule that participates in cell signaling pathways. As used herein,the term “glycolipid” refers to a carbohydrate-attached lipid thatserves as a marker for cellular recognition.

As used herein, the term “glycan” refers to a polysaccharide, oroligosaccharide. Glycan is also used herein to refer to the carbohydrateportion of a glycoconjugate, such as a glycoprotein, glycolipid,glycopeptide, glycoproteome, peptidoglycan, lipopolysaccharide or aproteoglycan. Glycans usually consist solely of O-glycosidic linkagesbetween monosaccharides. For example, cellulose is a glycan (or morespecifically a glucan) composed of beta-1,4-linked D-glucose, and chitinis a glycan composed of beta-1,4-linked N-acetyl-D-glucosamine. Glycanscan be homo or heteropolymers of monosaccharide residues, and can belinear or branched. Glycans can be found attached to proteins as inglycoproteins and proteoglycans. They are generally found on theexterior surface of cells. O- and N-linked glycans are very common ineukaryotes but may also be found, although less commonly, inprokaryotes. N-Linked glycans are found attached to the R-group nitrogen(N) of asparagine in the sequon. The sequon is a Asn-X-Ser or Asn-X-Thrsequence, where X is any amino acid except proline.

As used herein, the term “glycoprotein” refers to a protein covalentlymodified with glycan(s). There are four types of glycoproteins: 1)N-linked glycoproteins, 2) O-linked glycoproteins (mucins), 3)glucosaminoglycans (GAGs, which are also called proteoglycans), 4)GPI-anchored. Most glycoproteins have structural micro-heterogeneity(multiple different glycan structures attached within the sameglycosylation site), and structural macro-heterogeneity (multiple sitesand types of glycan attachment).

As used herein, the term “analog” refers to a compound, e.g., a drug,whose structure is related to that of another compound but whosechemical and biological properties may be quite different.

As used herein, the term “antigen” is defined as any substance capableof eliciting an immune response.

As used herein, the term “pathogen” is a biological agent that causesdisease or illness to it's host. The body contains many natural defensesagainst some of the common pathogens (such as Pneumocystis) in the formof the human immune system.

As used herein, the term “immunogen” refers to an antigen or a substancecapable of inducing production of an antigen, such as a DNA vaccine.

As used herein, the term “immunogenicity” refers to the ability of animmunogen, antigen, or vaccine to stimulate an immune response.

As used herein, the term “immunotherapy” refers to an array of treatmentstrategies based upon the concept of modulating the immune system toachieve a prophylactic and/or therapeutic goal.

Other Definitions

The terms “treating” and “treatment” as used herein refer to theadministration of an agent or formulation to a clinically symptomaticindividual afflicted with an adverse condition, disorder, or disease, soas to effect a reduction in severity and/or frequency of symptoms,eliminate the symptoms and/or their underlying cause, and/or facilitateimprovement or remediation of damage. The terms “preventing” and“prevention” refer to the administration of an agent or composition to aclinically asymptomatic individual who is susceptible to a particularadverse condition, disorder, or disease, and thus relates to theprevention of the occurrence of symptoms and/or their underlying cause.Unless otherwise indicated herein, either explicitly or by implication,if the term “treatment” (or “treating”) is used without reference topossible prevention, it is intended that prevention be encompassed aswell.

“Optional” or “optionally present”—as in an “optional substituent” or an“optionally present additive” means that the subsequently describedcomponent (e.g., substituent or additive) may or may not be present, sothat the description includes instances where the component is presentand instances where it is not.

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, e.g., the material may beincorporated into a formulation of the invention without causing anyundesirable biological effects or interacting in a deleterious mannerwith any of the other components of the dosage form formulation.However, when the term “pharmaceutically acceptable” is used to refer toa pharmaceutical excipient, it is implied that the excipient has met therequired standards of toxicological and manufacturing testing and/orthat it is included on the Inactive Ingredient Guide prepared by theU.S. Food and Drug Administration. As explained in further detail infra,“pharmacologically active” (or simply “active”) as in a“pharmacologically active” derivative or analog refers to derivative oranalog having the same type of pharmacological activity as the parentagent.

As used herein, the term “immunogen” refers to an antigen or a substancecapable of inducing production of an antigen, such as a DNA vaccine.

As used herein, the term “immunogenicity” refers to the ability of animmunogen, antigen, or vaccine to stimulate an immune response.

As used herein, the term “immunotherapy” refers to an array of treatmentstrategies based upon the concept of modulating the immune system toachieve a prophylactic and/or therapeutic goal.

As used herein, the term “cytokine” refers to any of numerous small,secreted proteins that regulate the intensity and duration of the immuneresponse by affecting immune cells differentiation process usuallyinvolving changes in gene expression by which a precursor cell becomes adistinct specialized cell type. Cytokines have been variously named aslymphokines, interleukins, and chemokines, based on their presumedfunction, cell of secretion, or target of action. For example, somecommon interleukins include, but are not limited to, IL-12, IL-18, IL-2,IFN-γ, TNF, IL-4, IL-10, IL-13, IL-21 and TGF-β. “Cytokine” is a genericterm for a group of proteins released by one cell population which acton another cell population as intercellular mediators. Examples of suchcytokines are lymphokines, monokines, and traditional polypeptidehormones. Included among the cytokines are interferons (IFN, notablyIFN-γ), interleukins (IL, notably IL-1, IL-2, IL-4, IL-10, IL-12),colony stimulating factors (CSF), thrombopoietin (TPO), erythropoietin(EPO), leukemia inhibitory factor (LIF), kit-ligand, growth hormones(GH), insulin-like growth factors (IGF), parathyroid hormone, thyroxine,insulin, relaxin, follicle stimulating hormone (FSH), thyroidstimulating hormone (TSH), leutinizing hormone (LH), hematopoieticgrowth factor, hepatic growth factor, fibroblast growth factors (FGF),prolactin, placental lactogen, tumor necrosis factors (TNF),mullerian-inhibiting substance, mouse gonadotropin-associated peptide,inhibin, activin, vascular endothelial growth factor (VEGF), integrin,nerve growth factors (NGF), platelet growth factor, transforming growthfactors (TGF), osteoinductive factors, etc.

As used herein, the term “chemokine” refers to any of various smallchemotactic cytokines released at the site of infection that provide ameans for mobilization and activation of lymphocytes. Chemokines attractleukocytes to infection sites. Chemokines have conserved cysteineresidues that allow them to be assigned to four groups. The groups, withrepresentative chemokines, are C—C chemokines (RANTES, MCP-1, MIP-1α,and MIP-1β), C—X—C chemokines (IL-8), C chemokines (Lymphotactin), andCXXXC chemokines (Fractalkine).

As used herein, the term “epitope” is defined as the parts of an antigenmolecule which contact the antigen binding site of an antibody or a Tcell receptor.

To further explain differential binding avidities of the ternary complexin mice and men, computer modeling was performed based on the x-raystructures of murine and human CD1d-αGalCer-iNKT TCR complexes,respectively (PDB access code 3HUJ, 3QUX, 3QUY, 3QUZ, and 3HE6).²⁷⁻²⁹(27) Borg, N. A.; Wun, K. S.; Kjer-Nielsen, L.; Wilce, M. C.; Pellicci,D. G.; Koh, R.; Besra, G. S.; Bharadwaj, M.; Godfrey, D. I.; McCluskey,J.; Rossjohn, J. Nature 2007, 448, 44; (28) Pellicci, D. G.; Patel, O.;Kjer-Nielsen, L.; Pang, S. S.; Sullivan, L. C.; Kyparissoudis, K.;Brooks, A. G.; Reid, H. H.; Gras, S.; Lucet, I. S.; Koh, R.; Smyth, M.J.; Mallevaey, T.; Matsuda, J. L.; Gapin, L.; McCluskey, J.; Godfrey, D.I.; Rossjohn, J. Immunity 2009, 31, 47; and (29) Aspeslagh, S.; Li, Y.;Yu, E. D.; Pauwels, N.; Trappeniers, M.; Girardi, E.; Decruy, T.; VanBeneden, K.; Venken, K.; Drennan, M.; Leybaert, L.; Wang, J.; Franck, R.W.; Van Calenbergh, S.; Zajonc, D. M.; Elewaut, D. EMBO J. 2011, 30,2294.

As used herein, the term “vaccine” refers to a preparation that containsan antigen, consisting of whole disease-causing organisms (killed orweakened) or components of such organisms, such as proteins, peptides,or polysaccharides, that is used to confer immunity against the diseasethat the organisms cause. Vaccine preparations can be natural, syntheticor derived by recombinant DNA technology.

As used herein, the terms “immunologic adjuvant” refers to a substanceused in conjunction with an immunogen which enhances or modifies theimmune response to the immunogen. Specifically, the terms “adjuvant” and“immunoadjuvant” are used interchangeably in the present invention andrefer to a compound or mixture that may be non-immunogenic whenadministered to a host alone, but that augments the host's immuneresponse to another antigen when administered conjointly with thatantigen. Adjuvant-mediated enhancement and/or extension of the durationof the immune response can be assessed by any method known in the artincluding without limitation one or more of the following: (i) anincrease in the number of antibodies produced in response toimmunization with the adjuvant/antigen combination versus those producedin response to immunization with the antigen alone; (ii) an increase inthe number of T cells recognizing the antigen or the adjuvant; and (iii)an increase in the level of one or more Type I cytokines.

Exemplary adjuvants of the invention comprise compounds which can berepresented by a general Formula 1.

Preferably, the exemplary adjuvant of the invention is pharmaceuticallyacceptable for use in humans.

The adjuvant of the invention can be administered as part of apharmaceutical or vaccine composition comprising an antigen or as aseparate formulation, which is administered conjointly with a secondcomposition containing an antigen. In any of these compositionsglycosphingolipids (GSLs) with α-glucose (α-Glc) can be combined withother adjuvants and/or excipients/carriers. These other adjuvantsinclude, but are not limited to, oil-emulsion and emulsifier-basedadjuvants such as complete Freund's adjuvant, incomplete Freund'sadjuvant, MF59, or SAF; mineral gels such as aluminum hydroxide (alum),aluminum phosphate or calcium phosphate; microbially-derived adjuvantssuch as cholera toxin (CT), pertussis toxin, Escherichia coliheat-labile toxin (LT), mutant toxins (e.g., LTK63 or LTR72), BacilleCalmette-Guerin (BCG), Corynebacterium parvum, DNA CpG motifs, muramyldipeptide, or monophosphoryl lipid A; particulate adjuvants such asimmunostimulatory complexes (ISCOMs), liposomes, biodegradablemicrospheres, or saponins (e.g., QS-21); cytokines such as IFN-γ, IL-2,IL-12 or GM-CSF; synthetic adjuvants such as nonionic block copolymers,muramyl peptide analogues (e.g.,N-acetyl-muramyl-L-threonyl-D-isoglutamine [thr-MDP],N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine,N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-[1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy]-ethylamine),polyphosphazenes, or synthetic polynucleotides, and surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,hydrocarbon emulsions, or keyhole limpet hemocyanins (KLH). Preferably,these additional adjuvants are also pharmaceutically acceptable for usein humans.

Within the meaning of the present invention, the term “conjointadministration or co-administration” is used to refer to administrationof an immune adjuvant and an antigen simultaneously in one composition,or simultaneously in different compositions, or sequentially. For thesequential administration to be considered “conjoint”, however, theantigen and adjuvant must be administered separated by a time intervalthat still permits the adjuvant to augment the immune response to theantigen. For example, when the antigen is a polypeptide, the antigen andadjuvant are administered on the same day, preferably within an hour ofeach other, and most preferably simultaneously. However, when nucleicacid is delivered to the subject and the polypeptide antigen isexpressed in the subject's cells, the adjuvant is administered within 24hours of nucleic acid administration, preferably within 6 hours.

As used herein, the term “immunogenic” means that an agent is capable ofeliciting a humoral or cellular immune response, and preferably both. Animmunogenic entity is also antigenic. An immunogenic composition is acomposition that elicits a humoral or cellular immune response, or both,when administered to an animal having an immune system.

The term “antigen” refers to any agent (e.g., protein, peptide,polysaccharide, glycoprotein, glycolipid, nucleic acid, or combinationthereof) that, when introduced into a host, animal or human, having animmune system (directly or upon expression as in, e.g., DNA vaccines),is recognized by the immune system of the host and is capable ofeliciting an immune response. As defined herein, the antigen-inducedimmune response can be humoral or cell-mediated, or both. An agent istermed “antigenic” when it is capable of specifically interacting withan antigen recognition molecule of the immune system, such as animmunoglobulin (antibody) or T cell antigen receptor (TCR). Within themeaning of the present invention, the antigens are preferably “surfaceantigens”, i.e., expressed naturally on the surface of a pathogen, orthe surface of an infected cell, or the surface of a tumor cell. Amolecule that is antigenic need not be itself immunogenic, i.e., capableof eliciting an immune response without an adjuvant or carrier. As usedherein, the term “antigen specific” refers to a property of a cellpopulation such that supply of a particular antigen, or a fragment ofthe antigen, results in specific cell characteristic.

The term “epitope” or “antigenic determinant” refers to any portion ofan antigen recognized either by B cells, or T cells, or both.Preferably, interaction of such epitope with an antigen recognition siteof an immunoglobulin (antibody) or T cell antigen receptor (TCR) leadsto the induction of antigen-specific immune response. T cells recognizeproteins only when they have been cleaved into smaller peptides and arepresented in a complex called the “major histocompatability complex(MHC)” located on another cell's surface. There are two classes of MHCcomplexes-class I and class II, and each class is made up of manydifferent alleles. Class I MHC complexes are found on virtually everycell and present peptides from proteins produced inside the cell. Thus,class I MHC complexes are useful for killing cells infected by virusesor cells which have become cancerous as the result of expression of anoncogene. T cells which have a protein called CD8 on their surface, bindspecifically to the MHC class I/peptide complexes via the T cellreceptor (TCR). This leads to cytolytic effector activities. Class IIMHC complexes are found only on antigen-presenting cells (APC) and areused to present peptides from circulating pathogens which have beenendocytosed by APCs. T cells which have a protein called CD4 bind to theMHC class II/peptide complexes via TCR. This leads to the synthesis ofspecific cytokines which stimulate an immune response. To be effectivelyrecognized by the immune system via MHC class I presentation, anantigenic polypeptide has to contain an epitope of at least about 8 to10 amino acids, while to be effectively recognized by the immune systemvia MHC class II presentation, an antigenic polypeptide has to containan epitope of at least about 13 to 25 amino acids. See, e.g.,Fundamental Immunology, 3 Edition, W. E. Paul ed., 1999,Lippincott-Raven Publ.

The term “species-specific” antigen refers to an antigen that is onlypresent in or derived from a particular species. Thus, the term“malaria-derived” or “malaria-specific” antigen refers to a natural(e.g., irradiated sporozoites) or synthetic (e.g., chemically producedmultiple antigen peptide [MAP] or recombinantly synthesized polypeptide)antigen comprising at least one epitope (B cell and/or T cell) derivedfrom any one of the proteins constituting plasmodium (said plasmodiumbeing without limitation P. falciparum, P. vivax, P. malariae, P. ovale,P. reichenowi, P. knowlesi, P. cynomolgi, P. brasilianum, P. yoelii, P.berghei, or P. chabaudi) and comprising at least 5-10 amino acidresidues. A preferred plasmodial protein for antigen generation iscircumsporozoite (CS) protein, however, other proteins can be also used,e.g., Thrombospondin Related Adhesion (Anonymous) protein (TRAP), alsocalled Sporozoite Surface Protein 2 (SSP2), LSA I, hsp70, SALSA, STARP,Hep17, MSA, RAP-1, RAP-2, etc.

The term “vaccine” refers to a composition (e.g., protein or vector suchas, e.g., an adenoviral vector, Sindbis virus vector, or pox virusvector) that can be used to elicit protective immunity in a recipient.It should be noted that to be effective, a vaccine of the invention canelicit immunity in a portion of the immunized population, as someindividuals may fail to mount a robust or protective immune response,or, in some cases, any immune response. This inability may stem from theindividual's genetic background or because of an immunodeficiencycondition (either acquired or congenital) or immunosuppression (e.g.,due to treatment with chemotherapy or use of immunosuppressive drugs,e.g., to prevent organ rejection or suppress an autoimmune condition).Vaccine efficacy can be established in animal models.

The term “DNA vaccine” is an informal term of art, and is used herein torefer to a vaccine delivered by means of a recombinant vector. Analternative, and more descriptive term used herein is “vector vaccine”(since some potential vectors, such as retroviruses and lentiviruses areRNA viruses, and since in some instances non-viral RNA instead of DNA isdelivered to cells through the vector). Generally, the vector isadministered in vivo, but ex vivo transduction of appropriate antigenpresenting cells, such as dendritic cells (DC), with administration ofthe transduced cells in vivo, is also contemplated.

The term “treat” is used herein to mean to relieve or alleviate at leastone symptom of a disease in a subject. Within the meaning of the presentinvention, the term “treat” may also mean to prolong the prepatency,i.e., the period between infection and clinical manifestation of adisease. Preferably, the disease is either infectious disease (e.g.,viral, bacterial, parasitic, or fungal) or malignancy (e.g., solid orblood tumors such as sarcomas, carcinomas, gliomas, blastomas,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,lymphoma, leukemia, melanoma, etc.).

The term “protect” is used herein to mean prevent or treat, or both, asappropriate, development or continuance of a disease in a subject.Within the meaning of the present invention, the disease can be selectedfrom the group consisting of infection (e.g., viral, bacterial,parasitic, or fungal) and/or malignancy (e.g., solid or blood tumorssuch as sarcomas, carcinomas, gliomas, blastomas, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, lymphoma, leukemia,melanoma, etc.). For example, according to the present invention, atherapeutic administration of a tumor-specific antigen conjointly withan adjuvant comprising exemplary agents of Formula 1 can enhance ananti-tumor immune response leading to slow-down in tumor growth andmetastasis or even tumor regression.

The term “protective immunity” refers to an immune response in a hostanimal (either active/acquired or passive/innate, or both) which leadsto inactivation and/or reduction in the load of said antigen and togeneration of long-lasting immunity (that is acquired, e.g., throughproduction of antibodies), which prevents or delays the development of adisease upon repeated exposure to the same or a related antigen. A“protective immune response” comprises a humoral (antibody) immunity orcellular immunity, or both, effective to, e.g., eliminate or reduce theload of a pathogen or infected cell (or produce any other measurablealleviation of the infection), or to reduce a tumor burden in animmunized (vaccinated) subject. Within the meaning of the presentinvention, protective immunity may be partial.

Immune systems are classified into two general systems, the “innate” or“natural” immune system and the “acquired” or “adaptive” immune system.It is thought that the innate immune system initially keeps theinfection under control, allowing time for the adaptive immune system todevelop an appropriate response. Recent studies have suggested that thevarious components of the innate immune system trigger and augment thecomponents of the adaptive immune system, including antigen-specific Band T lymphocytes (Fearon and Locksley, supra; Kos, 1998, Immunol. Res.,17: 303; Romagnani, 1992, Immunol. Today, 13: 379; Banchereau andSteinman, 1988, Nature, 392: 245).

The term “innate immunity” or “natural immunity” refers to innate immuneresponses that are not affected by prior contact with the antigen. Cellsof the innate immune system, including macrophages and dendritic cells(DC), take up foreign antigens through pattern recognition receptors,combine peptide fragments of these antigens with MHC class I and classII molecules, and stimulate naïve CD8⁺ and CD4⁺ T cells respectively(Banchereau and Steinman, supra; Holmskov et al., 1994, Immunol. Today,15: 67; Ulevitch and Tobias, 1995, Annu. Rev. Immunol., 13: 437).Professional antigen-presenting cells (APC) communicate with these Tcells leading to the differentiation of naïve CD4⁺ T cells into T-helper1 (Th1) or T-helper 2 (Th2) lymphocytes that mediate cellular andhumoral immunity, respectively (Trinchieri, 1995, Annu. Rev. Immunol.,13: 251; Howard and O'Garra, 1992, Immunol. Today, 13: 198; Abbas etal., 1996, Nature, 383: 787; Okamura et al., 1998, Adv. Immunol., 70:281; Mosmann and Sad, 1996, Immunol. Today, 17: 138; O'Garra, 1998,Immunity, 8: 275).

The term “acquired immunity” or “adaptive immunity” is used herein tomean active or passive, humoral or cellular immunity that is establishedduring the life of an animal, is specific for the inducing antigen, andis marked by an enhanced response on repeated encounters with saidantigen. A key feature of the T lymphocytes of the adaptive immunesystem is their ability to detect minute concentrations ofpathogen-derived peptides presented by MHC molecules on the cellsurface.

As used herein, the term “augment the immune response” means enhancingor extending the duration of the immune response, or both. When referredto a property of an agent (e.g., adjuvant), the term “[able to] augmentthe immunogenicity” refers to the ability to enhance the immunogenicityof an antigen or the ability to extend the duration of the immuneresponse to an antigen, or both.

The phrase “enhance immune response” within the meaning of the presentinvention refers to the property or process of increasing the scaleand/or efficiency of immunoreactivity to a given antigen, saidimmunoreactivity being either humoral or cellular immunity, or both. Animmune response is believed to be enhanced, if any measurable parameterof antigen-specific immunoreactivity (e.g., antibody titer, T cellproduction) is increased at least two-fold, preferably ten-fold, mostpreferably thirty-fold.

The term “therapeutically effective” applied to dose or amount refers tothat quantity of a compound or pharmaceutical composition or vaccinethat is sufficient to result in a desired activity upon administrationto a mammal in need thereof. As used herein with respect to adjuvant—andantigen-containing compositions or vaccines, the term “therapeuticallyeffective amount/dose” is used interchangeably with the term“immunogenically effective amount/dose” and refers to the amount/dose ofa compound (e.g., an antigen and/or an adjuvant comprisingglycosphingolipids (GSLs) with α-glucose (α-Glc) or pharmaceuticalcomposition or vaccine that is sufficient to produce an effective immuneresponse upon administration to a mammal.

The phrase “pharmaceutically acceptable”, as used in connection withcompositions of the invention, refers to molecular entities and otheringredients of such compositions that are physiologically tolerable anddo not typically produce untoward reactions when administered to ahuman. Preferably, as used herein, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in mammals, and more particularly inhumans.

The term “carrier” applied to pharmaceutical or vaccine compositions ofthe invention refers to a diluent, excipient, or vehicle with which acompound (e.g., an antigen and/or an adjuvant comprisingglycosphingolipids (GSLs) with α-glucose (α-Glc)) is administered. Suchpharmaceutical carriers can be sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.Water or aqueous solution, saline solutions, and aqueous dextrose andglycerol solutions are preferably employed as carriers, particularly forinjectable solutions. Suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin, 18^(th) Edition.

The term “native antibodies” or “immunoglobulins” refers to usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies between the heavy chainsof different immunoglobulin isotypes. Each heavy and light chain alsohas regularly spaced intrachain disulfide bridges. Each heavy chain hasat one end a variable domain (VH) followed by a number of constantdomains. Each light chain has a variable domain (VL) at one end and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light and heavy chain variable domains (Clothia etal., J Mol. Biol., 186: 651-663, 1985; Novotny and Haber, Proc. Natl.Acad. Sci. USA, 82: 4592-4596, 1985).

The term “antibody” or “Ab” is used in the broadest sense andspecifically covers not only native antibodies but also singlemonoclonal antibodies (including agonist and antagonist antibodies),antibody compositions with polyepitopic specificity, as well as antibodyfragments (e.g., Fab, F(ab′)2, scFv and Fv), so long as they exhibit thedesired biological activity.

As used herein, the term “CD1d” refers to a member of the CD1 (clusterof differentiation 1) family of glycoproteins expressed on the surfaceof various human antigen-presenting cells. CD1d presented lipid antigensactivate natural killer T cells. CD1d has a deep antigen-binding grooveinto which glycolipid antigens bind. CD1d molecules expressed ondendritic cells can bind and present glycolipids.

As used herein, the term “adaptive immune system” refers to highlyspecialized, systemic cells and processes that eliminate pathogenicchallenges. The cells of the adaptive immune system are a type ofleukocyte, called a lymphocyte. B cells and T cells are the major typesof lymphocytes.

As used herein, the term “T cells” and “Ts” refer to a group of whiteblood cells known as lymphocytes, that play a central role incell-mediated immunity. T cells can be distinguished from otherlymphocyte types, such as B cells and NKs by the presence of a specialreceptor on their cell surface called the T cell receptor (TCR). Severaldifferent subsets of T cells have been described, each with a distinctfunction. Helper T (T_(H)) Cells are the “middlemen” of the adaptiveimmune system. Once activated, they divide rapidly and secrete smallproteins called cytokines that regulate or “help” the immune response.Depending on the cytokine signals received, these cells differentiateinto T_(H)1, T_(H)2, T_(H)17, or one of other subsets, which secretedifferent cytokines.

As used herein, the term “antigen-presenting cell” (APC) refers to acell that displays foreign antigen complexed with majorhistocompatibility complex (MHC) on its surface. T-cells may recognizethis complex using their TCR. APCs fall into two categories:professional or non-professional. Dendritic cells (DCs) fall under theprofessional category and are capable of presenting antigen to T cells,in the context of CD1. In an exemplary implementation, the DCs utilizedin the methods of this disclosure may be of any of several DC subsets,which differentiate from, in one implementation, lymphoid or, in anotherimplementation, myeloid bone marrow progenitors.

As used herein, the term “naïve cell” refers to an undifferentiatedimmune system cell, for example a CD4 T-cell, that has not yetspecialized to recognize a specific pathogen.

As used herein, the term “natural killer cells” and “NKs” refers to aclass of lymphoid cells which are activated by interferons to contributeto innate host defense against viruses and other intracellularpathogens.

As used herein, the term “natural killer T cells” (NKTs) refers to asubset of T cells that share characteristics/receptors with bothconventional Ts and NKs. Many of these cells recognize thenon-polymorphic CD1d molecule, an antigen-presenting molecule that bindsself- and foreign lipids and glycolipids. The TCR of the NKTs are ableto recognize glycolipid antigens presented (chaperoned) by a CD1dmolecule. A major response of NKTs is rapid secretion of cytokines,including IL-4, IFN-γ and IL-10 after stimulation and thus influencediverse immune responses and pathogenic processes. The NKTs may be ahomogenous population or a heterogeneous population. In one exemplaryimplementation, the population may be “non-invariant NKTs”, which maycomprise human and mouse bone marrow and human liver T cell populationsthat are, for example, CD1d-reactive noninvariant T cells which expressdiverse TCRs, and which can also produce a large amount of IL-4 andIFN-γ. The best known subset of CD1d-dependent NKTs expresses aninvariant TCR-alpha (TCR-α) chain. These are referred to as type I orinvariant NKTs (iNKTs). These cells are conserved between humans (Vα24iNKTs) and mice (Vα14i NKTs) and are implicated in many immunologicalprocesses.

As used herein, the term “cytokine” refers to any of numerous small,secreted proteins that regulate the intensity and duration of the immuneresponse by affecting immune cells differentiation process usuallyinvolving changes in gene expression by which a precursor cell becomes adistinct specialized cell type. Cytokines have been variously named aslymphokines, interleukins, and chemokines, based on their presumedfunction, cell of secretion, or target of action. For example, somecommon interleukins include, but are not limited to, IL-12, IL-18, IL-2,IFN-γ, TNF, IL-4, IL-10, IL-13, IL-21 and TGF-β.

As used herein, the term “chemokine” refers to any of various smallchemotactic cytokines released at the site of infection that provide ameans for mobilization and activation of lymphocytes. Chemokines attractleukocytes to infection sites. Chemokines have conserved cysteineresidues that allow them to be assigned to four groups. The groups, withrepresentative chemokines, are C—C chemokines (RANTES, MCP-1, MIP-1α,and MIP-1β), C—X—C chemokines (IL-8), C chemokines (Lymphotactin), andCXXXC chemokines (Fractalkine).

As used herein, the term “T_(H)2-type response” refers to a pattern ofcytokine expression such that certain types of cytokines, interferons,chemokines are produced. Typical T_(H)2 cytokines include, but are notlimited to, IL-4, IL-5, IL-6 and IL-10.

As used herein, the term “T_(H)1-type response” refers to a pattern ofcytokine expression such that certain types of cytokines, interferons,chemokines are produced. Typical T_(H)1 cytokines include, but are notlimited to, IL-2, IFN-γ, GM-CSF and TNF-β.

As used herein, the term “T_(H)1 biased” refers to am immunogenicresponse in which production of T_(H)1 cytokines and/or chemokines isincreased to a greater extent than production of T_(H)2 cytokines and/orchemokines.

As used herein, the term “antimicrobial” refers to a substance thatkills or inhibits the growth of microbes such as bacteria, fungi, orviruses.

As used herein, the term “toxoid” refers to a bacterial toxin whosetoxicity has been weakened or suppressed either by chemical (formalin)or heat treatment, while other properties, typically immunogenicity, aremaintained. Toxoids are used in vaccines as they induce an immuneresponse to the original toxin or increase the response to anotherantigen. For example, the tetanus toxoid is derived from thetetanospasmin produced by Clostridium tetani and causing tetanus. Thetetanus toxoid is used by many plasma centers in the United States forthe development of plasma rich vaccines.

As used herein, the term “DNA vaccine” refers to a DNA construct that isintroduced into cells and subsequently translated into specificantigenic proteins.

As used herein, the term “plasmid” refers to an extrachromosomalcircular DNA capable of replicating, which may be used as a cloningvector.

As used herein, the term “microorganism” and “microbe” refers to anorganism that is microscopic (too small to be seen by the naked humaneye). Microorganisms are incredibly diverse and include, but are notlimited to, bacteria and fungi.

As used herein, the term “adjuvant or immunologic adjuvant” refers to asubstance used in conjunction with an immunogen which enhances ormodifies the immune response to the immunogen. In an exemplarycompound/analogs of the present disclosure are used as immunologicadjuvants to modify or augment the effects of a vaccine by stimulatingthe immune system of a patient who is administered the vaccine torespond to the vaccine more vigorously.

As used herein, the term “alum adjuvant” refers to an aluminum salt withimmune adjuvant activity. This agent adsorbs and precipitates proteinantigens in solution; the resulting precipitate improves vaccineimmunogenicity by facilitating the slow release of antigen from thevaccine depot formed at the site of inoculation.

As used herein, the term “anti-tumor immunotherapy active agent” refersto an exemplary compound/analog of the present disclosure that inhibits,reduces and/or eliminates tumors.

As used herein, the term “granulocyte-macrophage colony-stimulatingfactor” (GM-CSF) refers to a cytokine which serves as acolony-stimulating factor that stimulates production of white bloodcells, particularly granulocytes (neutrophils, basophils, andeosinophils), macrophages, and cells in the bone marrow that areprecursors of platelets.

As used herein, the term “antigen specific” refers to a property of acell population such that supply of a particular antigen, or a fragmentof the antigen, results in specific cell proliferation.

As used herein, the term “Flow cytometry” or “FACS” means a techniquefor examining the physical and chemical properties of particles or cellssuspended in a stream of fluid, through optical and electronic detectiondevices.

Amino acid residues in peptides shall hereinafter be abbreviated asfollows: P Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine isIle or I; Methionine is Met or M; Valine is Val or V; Serine is Ser orS; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A;Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gln or Q;Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D;Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W;Arginine is Arg or R; and Glycine is Gly or G. For further descriptionof amino acids, please refer to Proteins: Structure and MolecularProperties by Creighton, T. E., W. H. Freeman & Co., New York 1983.

Mammalian and mycobacterial lipids are known to be presented by humanCD1a, CD1b, CD1c, and CD1d. α-Galactosyl ceramide, a lipid found in themarine sponge Agelas mauritianus, has been the most extensively studiedligand for CD1d. It has been shown that in vitro stimulation of mousespleen cells by α-GalCer led to the proliferation of NKTs and productionof both IFN-□ and IL-4, a T_(H)1-type and T_(H)2-type response,respectively. Murine studies have shown that cells can be rapidlyactivated by immature dendritic cells (iDCs) bearing α-GalCer and thatthe activated iNKTs can in turn induce full maturation of DCs.

Uses of Adjuvants Comprising Glycosphingolipids (GSLs) with α-Glucose(α-Glc)

In one aspect, the present invention provides a method for augmenting animmunogenicity of an antigen in a mammal, comprising administering saidantigen conjointly with an adjuvant composition comprising aglycosphingolipids (GSLs) with α-glucose (α-Glc) of Formula 1. Accordingto the present invention, the use of glycosphingolipids (GSLs) withα-glucose (α-Glc) as an adjuvant results in an enhancement and/orextension of the duration of the protective immunity induced by theantigen. For example, as disclosed herein, conjoint administration ofglycosphingolipids (GSLs) with α-glucose (α-Glc) with peptidescorresponding to T cell or B cell epitopes of tumor or viral antigens,or DNA constructs expressing these antigens enhances antigen-specificimmune responses.

The glycosphingolipids (GSLs) with α-glucose (α-Glc)-containing adjuvantof the invention can be conjointly administered with any antigen, inparticular, with antigens derived from infectious agents or tumors.

The immunostimulating effects both in mice and humans may depend on theexpression of CD1d molecules and are mediated by NKT cells. Indeed, theinstant invention demonstrates that adjuvant activity is attributed atleast in part to its ability to enhance and/or extend NKT-mediatedantigen-specific Th1-type T cell responses and CD8+ T cell (or Tc)responses.

From an immunotherapy view point, glycosphingolipids (GSLs) withα-glucose (α-Glc) activation of the NKT cell system appears to havedistinct advantages over the other mechanisms for the following reasons:(a) the level of cytotoxicity of activated NKT cells is very high andeffective against a wide variety of tumor cells or infected cells; (b)the activation of NKT cells by glycosphingolipids (GSLs) with α-glucose(α-Glc) is totally dependent on a CD1d molecule, which is monomorphicamong individuals (Porcelli, Adv. Immunol., 59: 1-98, 1995), indicatingthat glycosphingolipids (GSLs) with α-glucose (α-Glc)-containingadjuvants can be utilized by all patients, regardless of MHC haplotype;(c) antigen-presenting functions of DC and NKT activation of humanpatients can be evaluated before immunotherapy by the in vivo assays inmice using Vα14 NKT cell status as an indicator.

According to the present invention, an adjuvant comprisingglycosphingolipids (GSLs) with α-glucose (α-Glc) of Formula 1 andantigen can be administered either as two separate formulations or aspart of the same composition. If administered separately, the adjuvantand antigen can be administered either sequentially or simultaneously.As disclosed herein, simultaneous administration of glycosphingolipids(GSLs) with α-glucose (α-Glc) adjuvant with the antigen is preferred andgenerally allows to achieve the most efficient immunostimulation.

As the glycosphingolipids (GSLs) with α-glucose (α-Glc) adjuvant of theinvention exerts its immunostimulatory activity in combination with aplurality of different antigens, it is therefore useful for bothpreventive and therapeutic applications. Accordingly, in a furtheraspect, the invention provides a prophylactic and/or therapeutic methodfor treating a disease in a mammal comprising conjointly administeringto said mammal an antigen and an adjuvant comprising aglycosphingolipids (GSLs) with α-glucose (α-Glc) of Formula 1. Thismethod can be useful, e.g., for protecting against and/or treatingvarious infections as well as for treating various neoplastic diseases.

Immunogenicity enhancing methods of the invention can be used to combatinfections, which include, but are not limited to, parasitic infections(such as those caused by plasmodial species, etc.), viral infections(such as those caused by influenza viruses, leukemia viruses,immunodeficiency viruses such as HIV, papilloma viruses, herpes virus,hepatitis viruses, measles virus, poxviruses, mumps virus,cytomegalovirus [CMV], Epstein-Barr virus, etc.), bacterial infections(such as those caused by staphylococcus, streptococcus, pneumococcus,Neisseria gonorrhea, Borrelia, pseudomonas, etc.), and fungal infections(such as those caused by candida, trichophyton, ptyrosporum, etc.).

Methods of the invention are also useful in treatment of variouscancers, which include without limitation fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendothelio-sarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, lymphoma, leukemia, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervicalcancer, testicular tumor, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

As further disclosed herein, maximal efficiency of the immunogenicityenhancing methods of present invention is attained when an antigen andglycosphingolipids (GSLs) with α-glucose (α-Glc) adjuvant areadministered simultaneously.

The methods of the invention can be used in conjunction with othertreatments. For example, an anti-cancer treatment using tumor-specificantigen and glycosphingolipids (GSLs) with α-glucose (α-Glc)-containingadjuvant of the present invention can be used in combination withchemotherapy and/or radiotherapy and/or IL-12 treatment. Anti-viralvaccines comprising glycosphingolipids (GSLs) with α-glucose(α-Glc)-containing adjuvant can be used in combination withIFN-αtreatment.

Glycosphingolipids (GSLs) with α-Glucose (α-Glc)-ContainingPharmaceutical and Vaccine Compositions

In conjunction with the methods of the present invention, also providedare pharmaceutical and vaccine compositions comprising animmunogenically effective amount of an antigen and immunogenicallyeffective amount of an adjuvant comprising glycosphingolipids (GSLs)with α-glucose (α-Glc) as well as, optionally, an additionalimmunostimulant, carrier or excipient (preferably all pharmaceuticallyacceptable). Said antigen and adjuvant can be either formulated as asingle composition or as two separate compositions, which can beadministered simultaneously or sequentially.

Adjuvants of the invention comprise compounds which belong to the classof sphingoglycolipids, specifically glycosphingolipids (GSLs) withα-glucose (α-Glc), which can be represented by a general Formula 1:

The antigens used in immunogenic (e.g., vaccine) compositions of theinstant invention can be derived from a eukaryotic cell (e.g., tumor,parasite, fungus), bacterial cell, viral particle, or any portionthereof (e.g. attenuated viral particles or viral components). In theevent the material to which the immunogenic response is to be directedis poorly antigenic, it may be additionally conjugated to a carriermolecule such as albumin or hapten, using standard covalent bindingtechniques, for example, with one of the several commercially availablereagent kits.

Examples of preferred tumor antigens of the present invention includetumor-specific proteins such as ErbB receptors, Melan A [MART1], gp100,tyrosinase, TRP-1/gp75, and TRP-2 (in melanoma); MAGE-1 and MAGE-3 (inbladder, head and neck, and non-small cell carcinoma); HPV EG and E7proteins (in cervical cancer); Mucin [MUC-1] (in breast, pancreas,colon, and prostate cancers); prostate-specific antigen [PSA] (inprostate cancer); carcinoembryonic antigen [CEA] (in colon, breast, andgastrointestinal cancers) and such shared tumor-specific antigens asMAGE-2, MAGE-4, MAGE-6, MAGE-10, MAGE-12, BAGE-1, CAGE-1,2,8, CAGE-3 to7, LAGE-1, NY-ESO-1/LAGE-2, NA-88, GnTV, and TRP2-INT2. The foregoinglist of antigens are intended as exemplary, as the antigen of interestcan be derived from any animal or human pathogen or tumor.

In a specific embodiment, the antigen of the invention may be presentedby a recombinant virus expressing said antigen. Preferably, the virus isselected from the group consisting of a recombinant adenovirus,recombinant pox virus, and recombinant Sindbis virus.

In the disclosed compositions, both the antigen and theglycosphingolipids (GSLs) with α-glucose (α-Glc) adjuvant are present inimmunogenically effective amounts. For each specific antigen, theoptimal immunogenically effective amount should be determinedexperimentally (taking into consideration specific characteristics of agiven patient and/or type of treatment). Generally, this amount is inthe range of 0.1 μg-100 mg of an antigen per kg of the body weight. Forthe glycosphingolipids (GSLs) with α-glucose (α-Glc) adjuvant of thepresent invention, the optimal immunogenically effective amount ispreferably in the range of 10-100 μg of the adjuvant per kg of the bodyweight.

The invention also provides a method for preparing a vaccine compositioncomprising at least one antigen and an adjuvant comprisingglycosphingolipids (GSLs) with α-glucose (α-Glc) of Formula 1, saidmethod comprising admixing the adjuvant and the antigen, and optionallyone or more physiologically acceptable carriers and/or excipients and/orauxiliary substances.

Formulations and Administration

The invention provides pharmaceutical and vaccine formulationscontaining therapeutics of the invention (an antigen andglycosphingolipids (GSLs) with α-glucose (α-Glc) adjuvant either as asingle composition or as two separate compositions which can beadministered simultaneously or sequentially), which formulations aresuitable for administration to elicit an antigen-specific protectiveimmune response for the treatment and prevention of infectious orneoplastic diseases described above. Compositions of the presentinvention can be formulated in any conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, an antigenand/or an adjuvant comprising a glycosphingolipids (GSLs) with α-glucose(α-Glc) of Formula 1, can be formulated for administration bytransdermal delivery, or by transmucosal administration, including butnot limited to, oral, buccal, intranasal, ophthalmic, vaginal, rectal,intracerebral, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous routes, via scarification (scratching through the toplayers of skin, e.g., using a bifurcated needle), by inhalation(pulmonary) or insufflation (either through the mouth or the nose), orby administration to antigen presenting cells ex vivo followed byadministration of the cells to the subject, or by any other standardroute of immunization.

Preferably, the immunogenic formulations of the invention can bedelivered parenterally, i.e., by intravenous (i.v.), subcutaneous(s.c.), intraperitoneal (i.p.), intramuscular (i.m.), subdermal (s.d.),or intradermal (i.d.) administration, by direct injection, via, forexample, bolus injection, continuous infusion, or gene gun (e.g., toadminister a vector vaccine to a subject, such as naked DNA or RNA).Formulations for injection can be presented in unit dosage form, e.g.,in ampoules or in multi-dose containers, with an added preservative. Thecompositions can take such forms as excipients, suspensions, solutionsor emulsions in oily or aqueous vehicles, and can contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.Alternatively, the active ingredient can be in powder form forreconstitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use.

The present invention also contemplates various mucosal vaccinationstrategies. While the mucosa can be targeted by local delivery of avaccine, various strategies have been employed to deliver immunogeniccompositions to the mucosa. For example, in a specific embodiment, theimmunogenic polypeptide or vector vaccine can be administered in anadmixture with, or as a conjugate or chimeric fusion protein with,cholera toxin, such as cholera toxin B or a cholera toxin A/B chimera(see, e.g., Hajishengallis, J Immunol., 154: 4322-32, 1995; Jobling andHolmes, Infect Immun., 60: 4915-24, 1992; Lebens and Holmgren, Dev BiolStand 82:215-27, 1994). In another embodiment, an admixture with heatlabile enterotoxin (LT) can be prepared for mucosal vaccination. Othermucosal immunization strategies include encapsulating the immunogen inmicrocapsules (see, e.g., U.S. Pat. Nos. 5,075,109; 5,820,883, and5,853,763) and using an immunopotentiating membranous carrier (see,e.g., PCT Application No. WO 98/0558). Immunogenicity of orallyadministered immunogens can be enhanced by using red blood cells (rbc)or rbc ghosts (see, e.g., U.S. Pat. No. 5,643,577), or by using bluetongue antigen (see, e.g., U.S. Pat. No. 5,690,938). Systemicadministration of a targeted immunogen can also produce mucosalimmunization (see, U.S. Pat. No. 5,518,725). Various strategies can bealso used to deliver genes for expression in mucosal tissues, such asusing chimeric rhinoviruses (see, e.g., U.S. Pat. No. 5,714,374),adenoviruses, vaccinia viruses, or specific targeting of a nucleic acid(see, e.g., PCT Application No. WO 97/05267).

For oral administration, the formulations of the invention can take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinized maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets can be coated by methods well known in the art. The compositionsof the invention can be also introduced in microspheres ormicrocapsules, e.g., fabricated from poly-glycolic acid/lactic acid(PGLA) (see, U.S. Pat. Nos. 5,814,344; 5,100,669 and 4,849,222; PCTPublication Nos. WO 95/11010 and WO 93/07861). Liquid preparations fororal administration can take the form of, for example, solutions,syrups, emulsions or suspensions, or they can be presented as a dryproduct for reconstitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations can also contain buffer salts, flavoring,coloring and sweetening agents as appropriate. Preparations for oraladministration can be suitably formulated to give controlled release ofthe active compound.

For administration by inhalation, the therapeutics according to thepresent invention can be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoro-methane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitcan be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator can be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

Compositions of the present invention can also be formulated in rectalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compositionscan also be formulated as a depot preparation. Such long actingformulations can be administered by implantation (for example,subcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds can be formulated with suitable polymeric orhydrophobic materials (for example, as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

As disclosed herein, an antigen and/or glycosphingolipids (GSLs) withα-glucose (α-Glc) adjuvant can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredients.Suitable excipients are, for example, water, saline, buffered saline,dextrose, glycerol, ethanol, sterile isotonic aqueous buffer or the likeand combinations thereof. In addition, if desired, the preparations mayalso include minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents, and/or immune stimulators(e.g., adjuvants in addition to glycosphingolipids (GSLs) with α-glucose(α-Glc)) that enhance the effectiveness of the pharmaceuticalcomposition or vaccine. Non-limiting examples of additional immunestimulators which may enhance the effectiveness of the compositions ofthe present invention include immunostimulatory, immunopotentiating, orpro-inflammatory cytokines, lymphokines, or chemokines or nucleic acidsencoding them (specific examples include interleukin (IL)-1, IL-2, IL-3,IL-4, IL-12, IL-13, granulocyte-macrophage (GM)-colony stimulatingfactor (CSF) and other colony stimulating factors, macrophageinflammatory factor, Flt3 ligand, see additional examples ofimmunostimulatory cytokines in the Section entitled “Definitions”).These additional immunostimulatory molecules can be deliveredsystemically or locally as proteins or by expression of a vector thatcodes for expression of the molecule. The techniques described above fordelivery of the antigen and glycosphingolipids (GSLs) with α-glucose(α-Glc) adjuvant can also be employed for the delivery of additionalimmunostimulatory molecules.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of theimmunogenic formulations of the invention. In a related embodiment, thepresent invention provides a kit for the preparation of a pharmaceuticalor vaccine composition comprising at least one antigen and aglycosphingolipids (GSLs) with α-glucose (α-Glc)-containing adjuvant,said kit comprising the antigen in a first container, and the adjuvantin a second container, and optionally instructions for admixing theantigen and the adjuvant and/or for administration of the composition.Each container of the kit may also optionally include one or morephysiologically acceptable carriers and/or excipients and/or auxiliarysubstances. Associated with such container(s) can be a notice in theform prescribed by a governmental agency regulating the manufacture, useor sale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient (i.e., an antigen and/or a glycosphingolipids (GSLs)with α-glucose (α-Glc)-containing adjuvant). The pack may, for example,comprise metal or plastic foil, such as a blister pack. The pack ordispenser device may be accompanied by instructions for administration.Compositions of the invention formulated in a compatible pharmaceuticalcarrier may also be prepared, placed in an appropriate container, andlabeled for treatment of an indicated condition.

Effective Dose and Safety Evaluations

According to the methods of the present invention, the pharmaceuticaland vaccine compositions described herein are administered to a patientat immunogenically effective doses, preferably, with minimal toxicity.As recited in the Section entitled “Definitions”, “immunogenicallyeffective dose” or “therapeutically effective dose” of disclosedformulations refers to that amount of an antigen and/orglycosphingolipids (GSLs) with α-glucose (α-Glc) adjuvant that issufficient to produce an effective immune response in the treatedsubject and therefore sufficient to result in a healthful benefit tosaid subject.

Following methodologies which are well-established in the art (see,e.g., reports on evaluation of several vaccine formulations containingnovel adjuvants in a collaborative effort between the Center forBiological Evaluation and Food and Drug Administration and the NationalInstitute of Allergy and Infectious Diseases [Goldenthal et al.,National Cooperative Vaccine Development Working Group. AIDS Res. Hum.Retroviruses, 1993, 9:545-9]), effective doses and toxicity of thecompounds and compositions of the instant invention are first determinedin preclinical studies using small animal models (e.g., mice) in whichboth the antigen and glycosphingolipids (GSLs) with α-glucose(α-Glc)-containing adjuvant has been found to be immunogenic and thatcan be reproducibly immunized by the same route proposed for the humanclinical trials. Specifically, for any pharmaceutical composition orvaccine used in the methods of the invention, the therapeuticallyeffective dose can be estimated initially from animal models to achievea circulating plasma concentration range that includes the IC₅₀ (i.e.,the concentration of the test compound which achieves a half-maximalinhibition of symptoms). Dose-response curves derived from animalsystems are then used to determine testing doses for the initialclinical studies in humans. In safety determinations for eachcomposition, the dose and frequency of immunization should meet orexceed those anticipated for use in the clinical trial.

As disclosed herein, the dose of glycosphingolipids (GSLs) withα-glucose (α-Glc), antigen(s) and other components in the compositionsof the present invention is determined to ensure that the doseadministered continuously or intermittently will not exceed a certainamount in consideration of the results in test animals and theindividual conditions of a patient. A specific dose naturally variesdepending on the dosage procedure, the conditions of a patient or asubject animal such as age, body weight, sex, sensitivity, feed, dosageperiod, drugs used in combination, seriousness of the disease. Theappropriate dose and dosage times under certain conditions can bedetermined by the test based on the above-described indices and shouldbe decided according to the judgment of the practitioner and eachpatient's circumstances according to standard clinical techniques. Inthis connection, the dose of an antigen is generally in the range of 0.1μg-100 mg per kg of the body weight, and the dose of theglycosphingolipids (GSLs) with α-glucose (α-Glc) adjuvant required foraugmenting the immune response to the antigen is generally in the rangeof 10-100 μg per kg of the body weight.

Toxicity and therapeutic efficacy of glycosphingolipids (GSLs) withα-glucose (α-Glc)-containing immunogenic compositions of the inventioncan be determined by standard pharmaceutical procedures in experimentalanimals, e.g., by determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅/ED₅₀.Compositions that exhibit large therapeutic indices are preferred. Whiletherapeutics that exhibit toxic side effects can be used (e.g., whentreating severe forms of cancer or life-threatening infections), careshould be taken to design a delivery system that targets suchimmunogenic compositions to the specific site (e.g., lymphoid tissuemediating an immune response, tumor or an organ supporting replicationof the infectious agent) in order to minimize potential damage to othertissues and organs and, thereby, reduce side effects. As disclosedherein (see also Background Section and Examples), theglycosphingolipids (GSLs) with α-glucose (α-Glc) adjuvant of theinvention is not only highly immunostimulating at relatively low doses(e.g., 10-100 μg of the adjuvant per kg of the body weight) but alsopossesses low toxicity and does not produce significant side effects.

As specified above, the data obtained from the animal studies can beused in formulating a range of dosage for use in humans. Thetherapeutically effective dosage of glycosphingolipids (GSLs) withα-glucose (α-Glc)-containing compositions of the present invention inhumans lies preferably within a range of circulating concentrations thatinclude the ED₅₀ with little or no toxicity. The dosage can vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. Ideally, a single dose should be used.

Definitions Directed to Chemical Structures

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in ThomasSorrell, Organic Chemistry, University Science Books, Sausalito, 1999;Smith and March, March's Advanced Organic Chemistry, 5^(th) Edition,John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various isomeric forms, e.g., enantiomers and/ordiastereomers. For example, the compounds described herein can be in theform of an individual enantiomer, diastereomer or geometric isomer, orcan be in the form of a mixture of stereoisomers, including racemicmixtures and mixtures enriched in one or more stereoisomer. Isomers canbe isolated from mixtures by methods known to those skilled in the art,including for example, chiral high pressure liquid chromatography (HPLC)and the formation and crystallization of chiral salts; or preferredisomers can be prepared by asymmetric syntheses. See, for example,Jacques et al., Enantiomers, Racemates and Resolutions (WileyInterscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977);Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); andWilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E. L.Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind. 1972). Theinstant disclosure additionally encompasses compounds described hereinas individual isomers substantially free of other isomers, andalternatively, as mixtures of various isomers.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example, “C₁₋₆” is intended toencompass C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆.

“Alkyl” refers to a radical of a straight-chain or branched saturatedhydrocarbon group having from 1 to 20 carbon atoms (“C₁₋₂₀ alkyl”). Insome embodiments, an alkyl group has 1 to 10 carbon atoms (“C₁₋₁₀alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms(“C₁₋₉ alkyl”). In some embodiments, an alkyl group has 1 to 8 carbonatoms (“C₁₋₈ alkyl”). In some embodiments, an alkyl group has 1 to 7carbon atoms (“C₁₋₇ alkyl”). In some embodiments, an alkyl group has 1to 6 carbon atoms (“C₁₋₆ alkyl”). In some embodiments, an alkyl grouphas 1 to 5 carbon atoms (“C₁₋₅ alkyl”). In some embodiments, an alkylgroup has 1 to 4 carbon atoms (“C₁₋₄ alkyl”). In some embodiments, analkyl group has 1 to 3 carbon atoms (“C₁₋₃ alkyl”). In some embodiments,an alkyl group has 1 to 2 carbon atoms (“C₁₋₂ alkyl”). In someembodiments, an alkyl group has 1 carbon atom (“C₁ alkyl”). In someembodiments, an alkyl group has 2 to 6 carbon atoms (“C₂₋₆ alkyl”).Examples of C₁₋₆ alkyl groups include methyl (C₁), ethyl (C₂), n-propyl(C₃), iso-propyl (C₃), n-butyl (C₄), tert-butyl (C₄), sec-butyl (C₄),iso-butyl (C₄), n-pentyl (C₅), 3-pentanyl (C₅), amyl (C₅), neopentyl(C₅), 3-methyl-2-butanyl (C₅), tertiary amyl (C₅), and n-hexyl (C₆).Additional examples of alkyl groups include n-heptyl (C₇), n-octyl (C₈)and the like. Unless otherwise specified, each instance of an alkylgroup is independently optionally substituted, i.e., unsubstituted (an“unsubstituted alkyl”) or substituted (a “substituted alkyl”) with oneor more substituents. In certain embodiments, the alkyl group isunsubstituted C₁₋₁₀ alkyl (e.g., —CH₃). In certain embodiments, thealkyl group is substituted C₁₋₁₀ alkyl.

“Alkenyl” refers to a radical of a straight-chain or branchedhydrocarbon group having from 2 to 20 carbon atoms, one or morecarbon-carbon double bonds, and no triple bonds (“C₂₋₂₀ alkenyl”). Insome embodiments, an alkenyl group has 2 to 10 carbon atoms (“C₂₋₁₀alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms(“C₂₋₉ alkenyl”). In some embodiments, an alkenyl group has 2 to 8carbon atoms (“C₂₋₈ alkenyl”). In some embodiments, an alkenyl group has2 to 7 carbon atoms (“C₂₋₇ alkenyl”). In some embodiments, an alkenylgroup has 2 to 6 carbon atoms (“C₂₋₆ alkenyl”). In some embodiments, analkenyl group has 2 to 5 carbon atoms (“C₂₋₅ alkenyl”). In someembodiments, an alkenyl group has 2 to 4 carbon atoms (“C₂₋₄ alkenyl”).In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C₂₋₃alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C₂alkenyl”). The one or more carbon-carbon double bonds can be internal(such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples ofC₂₋₄ alkenyl groups include ethenyl (C₂), 1-propenyl (C₃), 2-propenyl(C₃), 1-butenyl (C₄), 2-butenyl (C₄), butadienyl (C₄), and the like.Examples of C₂₋₆ alkenyl groups include the aforementioned C₂₋₄ alkenylgroups as well as pentenyl (C₅), pentadienyl (C₅), hexenyl (C₆), and thelike. Additional examples of alkenyl include heptenyl (C₇), octenyl(C₈), octatrienyl (C₈), and the like. Unless otherwise specified, eachinstance of an alkenyl group is independently optionally substituted,i.e., unsubstituted (an “unsubstituted alkenyl”) or substituted (a“substituted alkenyl”) with one or more substituents. In certainembodiments, the alkenyl group is unsubstituted C₂₋₁₀ alkenyl. Incertain embodiments, the alkenyl group is substituted C₂₋₁₀ alkenyl.

“Alkynyl” refers to a radical of a straight-chain or branchedhydrocarbon group having from 2 to 20 carbon atoms, one or morecarbon-carbon triple bonds, and optionally one or more double bonds(“C₂₋₂₀ alkynyl”). In some embodiments, an alkynyl group has 2 to 10carbon atoms (“C₂₋₁₀ alkynyl”). In some embodiments, an alkynyl grouphas 2 to 9 carbon atoms (“C₂₋₉ alkynyl”). In some embodiments, analkynyl group has 2 to 8 carbon atoms (“C₂₋₈ alkynyl”). In someembodiments, an alkynyl group has 2 to 7 carbon atoms (“C₂₋₇ alkynyl”).In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C₂₋₆alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms(“C₂₋₅ alkynyl”). In some embodiments, an alkynyl group has 2 to 4carbon atoms (“C₂₋₄ alkynyl”). In some embodiments, an alkynyl group has2 to 3 carbon atoms (“C₂₋₃ alkynyl”). In some embodiments, an alkynylgroup has 2 carbon atoms (“C₂ alkynyl”). The one or more carbon-carbontriple bonds can be internal (such as in 2-butynyl) or terminal (such asin 1-butynyl). Examples of C₂₋₄ alkynyl groups include, withoutlimitation, ethynyl (C₂), 1-propynyl (C₃), 2-propynyl (C₃), 1-butynyl(C₄), 2-butynyl (C₄), and the like. Examples of C₂₋₆ alkenyl groupsinclude the aforementioned C₂₋₄ alkynyl groups as well as pentynyl (C₅),hexynyl (C₆), and the like. Additional examples of alkynyl includeheptynyl (C₇), octynyl (C₈), and the like. Unless otherwise specified,each instance of an alkynyl group is independently optionallysubstituted, i.e., unsubstituted (an “unsubstituted alkynyl”) orsubstituted (a “substituted alkynyl”) with one or more substituents. Incertain embodiments, the alkynyl group is unsubstituted C₂₋₁₀ alkynyl.In certain embodiments, the alkynyl group is substituted C₂₋₁₀ alkynyl.

“Carbocyclyl” or “carbocyclic” refers to a radical of a non-aromaticcyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C₃₋₁₀carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. Insome embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms(“C₃₋₈ carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to6 ring carbon atoms (“C₃₋₆ carbocyclyl”). In some embodiments, acarbocyclyl group has 3 to 6 ring carbon atoms (“C₃₋₆ carbocyclyl”). Insome embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms(“C₅₋₁₀ carbocyclyl”). Exemplary C₃₋₆ carbocyclyl groups include,without limitation, cyclopropyl (C₃), cyclopropenyl (C₃), cyclobutyl(C₄), cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl (C₅),cyclohexyl (C₆), cyclohexenyl (C₆), cyclohexadienyl (C₆), and the like.Exemplary C₃₋₈ carbocyclyl groups include, without limitation, theaforementioned C₃₋₆ carbocyclyl groups as well as cycloheptyl (C₇),cycloheptenyl (C₇), cycloheptadienyl (C₇), cycloheptatrienyl (C₇),cyclooctyl (C₈), cyclooctenyl (C₈), 40ydroxy[2.2.1]heptanyl (C₇),40ydroxy[2.2.2]octanyl (C₈), and the like. Exemplary C₃₋₁₀ carbocyclylgroups include, without limitation, the aforementioned C₃₋₈ carbocyclylgroups as well as cyclononyl (C₉), cyclononenyl (C₉), cyclodecyl (C₁₀),cyclodecenyl (C₁₀), octahydro-1H-indenyl (C₉), decahydronaphthalenyl(C₁₀), spiro[4.5]decanyl (C₁₀), and the like. As the foregoing examplesillustrate, in certain embodiments, the carbocyclyl group is eithermonocyclic (“monocyclic carbocyclyl”) or contain a fused, bridged orspiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) andcan be saturated or can be partially unsaturated. “Carbocyclyl” alsoincludes ring systems wherein the carbocyclic ring, as defined above, isfused to one or more aryl or heteroaryl groups wherein the point ofattachment is on the carbocyclic ring, and in such instances, the numberof carbons continue to designate the number of carbons in thecarbocyclic ring system. Unless otherwise specified, each instance of acarbocyclyl group is independently optionally substituted, i.e.,unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a“substituted carbocyclyl”) with one or more substituents. In certainembodiments, the carbocyclyl group is unsubstituted C₃₋₁₀ carbocyclyl.In certain embodiments, the carbocyclyl group is a substituted C₃₋₁₀carbocyclyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturatedcarbocyclyl group having from 3 to 10 ring carbon atoms (“C₃₋₁₀cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ringcarbon atoms (“C₃₋₈ cycloalkyl”). In some embodiments, a cycloalkylgroup has 3 to 6 ring carbon atoms (“C₃₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C₅₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ringcarbon atoms (“C₅₋₁₀ cycloalkyl”). Examples of C₅₋₆ cycloalkyl groupsinclude cyclopentyl (C₅) and cyclohexyl (C₅). Examples of C₃₋₆cycloalkyl groups include the aforementioned C₅₋₆ cycloalkyl groups aswell as cyclopropyl (C₃) and cyclobutyl (C₄). Examples of C₃₋₈cycloalkyl groups include the aforementioned C₃₋₆ cycloalkyl groups aswell as cycloheptyl (C₇) and cyclooctyl (C₈). Unless otherwisespecified, each instance of a cycloalkyl group is independentlyunsubstituted (an “unsubstituted cycloalkyl”) or substituted (a“substituted cycloalkyl”) with one or more substituents. In certainembodiments, the cycloalkyl group is unsubstituted C₃₋₁₀ cycloalkyl. Incertain embodiments, the cycloalkyl group is substituted C₃₋₁₀cycloalkyl.

“Heterocyclyl” or “heterocyclic” refers to a radical of a 3- to10-membered non-aromatic ring system having ring carbon atoms and 1 to 4ring heteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3-10 memberedheterocyclyl”). In certain embodiments, the heteroatom is independentlyselected from nitrogen, sulfur, and oxygen. In heterocyclyl groups thatcontain one or more nitrogen atoms, the point of attachment can be acarbon or nitrogen atom, as valency permits. A heterocyclyl group caneither be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged orspiro ring system such as a bicyclic system (“bicyclic heterocyclyl”),and can be saturated or partially unsaturated. Heterocyclyl bicyclicring systems can include one or more heteroatoms in one or both rings.“Heterocyclyl” also includes ring systems wherein the heterocyclic ring,as defined above, is fused with one or more carbocyclyl groups whereinthe point of attachment is either on the carbocyclyl or heterocyclicring, or ring systems wherein the heterocyclic ring, as defined above,is fused with one or more aryl or heteroaryl groups, wherein the pointof attachment is on the heterocyclic ring, and in such instances, thenumber of ring members continue to designate the number of ring membersin the heterocyclic ring system. Unless otherwise specified, eachinstance of heterocyclyl is independently optionally substituted, i.e.,unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a“substituted heterocyclyl”) with one or more substituents. In certainembodiments, the heterocyclyl group is unsubstituted 3-10 memberedheterocyclyl. In certain embodiments, the heterocyclyl group issubstituted 3-10 membered heterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 memberednon-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“5-10 memberedheterocyclyl”). In some embodiments, a heterocyclyl group is a 5-8membered non-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”). In someembodiments, a heterocyclyl group is a 5-6 membered non-aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms, wherein eachheteroatom is independently selected from nitrogen, oxygen, and sulfur(“5-6 membered heterocyclyl”). In some embodiments, the 5-6 memberedheterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen,and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2ring heteroatoms selected from nitrogen, oxygen, and sulfur. In someembodiments, the 5-6 membered heterocyclyl has one ring heteroatomselected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing one heteroatominclude, without limitation, azirdinyl, oxiranyl, and thiorenyl.Exemplary 4-membered heterocyclyl groups containing one heteroatominclude, without limitation, azetidinyl, oxetanyl, and thietanyl.Exemplary 5-membered heterocyclyl groups containing one heteroatominclude, without limitation, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl,and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groupscontaining two heteroatoms include, without limitation, dioxolanyl,oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-memberedheterocyclyl groups containing three heteroatoms include, withoutlimitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary6-membered heterocyclyl groups containing one heteroatom include,without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl,and thianyl. Exemplary 6-membered heterocyclyl groups containing twoheteroatoms include, without limitation, piperazinyl, morpholinyl,dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groupscontaining two heteroatoms include, without limitation, triazinanyl.Exemplary 7-membered heterocyclyl groups containing one heteroatominclude, without limitation, azepanyl, oxepanyl, and thiepanyl.Exemplary 8-membered heterocyclyl groups containing one heteroatominclude, without limitation, azocanyl, oxecanyl, and thiocanyl.Exemplary 5-membered heterocyclyl groups fused to a C₆ aryl ring (alsoreferred to herein as a 5,6-bicyclic heterocyclic ring) include, withoutlimitation, indolinyl, isoindolinyl, dihydrobenzofuranyl,dihydrobenzothienyl, benzoxazolinonyl, and the like. Exemplary6-membered heterocyclyl groups fused to an aryl ring (also referred toherein as a 6,6-bicyclic heterocyclic ring) include, without limitation,tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

“Aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclicor tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 itelectrons shared in a cyclic array) having 6-14 ring carbon atoms andzero heteroatoms in the aromatic ring system (“C₆₋₁₄ aryl”). In someembodiments, an aryl group has six ring carbon atoms (“C₆ aryl”; e.g.,phenyl). In some embodiments, an aryl group has ten ring carbon atoms(“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In someembodiments, an aryl group has fourteen ring carbon atoms (“C₁₄ aryl”;e.g., anthracyl). “Aryl” also includes ring systems wherein the arylring, as defined above, is fused with one or more carbocyclyl orheterocyclyl groups wherein the radical or point of attachment is on thearyl ring, and in such instances, the number of carbon atoms continue todesignate the number of carbon atoms in the aryl ring system. Unlessotherwise specified, each instance of an aryl group is independentlyoptionally substituted, i.e., unsubstituted (an “unsubstituted aryl”) orsubstituted (a “substituted aryl”) with one or more substituents. Incertain embodiments, the aryl group is unsubstituted C₆₋₁₄ aryl. Incertain embodiments, the aryl group is substituted C₆₋₁₄ aryl.

“Arylalkyl” is a subset of alkyl and aryl, as defined herein, and refersto an optionally substituted alkyl group substituted by an optionallysubstituted aryl group. In certain embodiments, the aralkyl isoptionally substituted benzyl. In certain embodiments, the aralkyl isbenzyl. In certain embodiments, the aralkyl is optionally substitutedphenethyl. In certain embodiments, the aralkyl is phenethyl.

“Heteroaryl” refers to a radical of a 5-10 membered monocyclic orbicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electronsshared in a cyclic array) having ring carbon atoms and 1-4 ringheteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from nitrogen, oxygen, and sulfur(“5-10 membered heteroaryl”). In heteroaryl groups that contain one ormore nitrogen atoms, the point of attachment can be a carbon or nitrogenatom, as valency permits. Heteroaryl bicyclic ring systems can includeone or more heteroatoms in one or both rings. “Heteroaryl” includes ringsystems wherein the heteroaryl ring, as defined above, is fused with oneor more carbocyclyl or heterocyclyl groups wherein the point ofattachment is on the heteroaryl ring, and in such instances, the numberof ring members continue to designate the number of ring members in theheteroaryl ring system. “Heteroaryl” also includes ring systems whereinthe heteroaryl ring, as defined above, is fused with one or more arylgroups wherein the point of attachment is either on the aryl orheteroaryl ring, and in such instances, the number of ring membersdesignates the number of ring members in the fused (aryl/heteroaryl)ring system. Bicyclic heteroaryl groups wherein one ring does notcontain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and thelike) the point of attachment can be on either ring, i.e., either thering bearing a heteroatom (e.g., 2-indolyl) or the ring that does notcontain a heteroatom (e.g., 5-indolyl).

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-8 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unlessotherwise specified, each instance of a heteroaryl group isindependently optionally substituted, i.e., unsubstituted (an“unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”)with one or more substituents. In certain embodiments, the heteroarylgroup is unsubstituted 5-14 membered heteroaryl. In certain embodiments,the heteroaryl group is substituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing one heteroatominclude, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary5-membered heteroaryl groups containing two heteroatoms include, withoutlimitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, andisothiazolyl. Exemplary 5-membered heteroaryl groups containing threeheteroatoms include, without limitation, triazolyl, oxadiazolyl, andthiadiazolyl. Exemplary 5-membered heteroaryl groups containing fourheteroatoms include, without limitation, tetrazolyl. Exemplary6-membered heteroaryl groups containing one heteroatom include, withoutlimitation, pyridinyl. Exemplary 6-membered heteroaryl groups containingtwo heteroatoms include, without limitation, pyridazinyl, pyrimidinyl,and pyrazinyl. Exemplary 6-membered heteroaryl groups containing threeor four heteroatoms include, without limitation, triazinyl andtetrazinyl, respectively. Exemplary 7-membered heteroaryl groupscontaining one heteroatom include, without limitation, azepinyl,oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groupsinclude, without limitation, indolyl, isoindolyl, indazolyl,benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl,benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl,benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl,indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groupsinclude, without limitation, naphthyridinyl, pteridinyl, quinolinyl,isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

“Heteroaralkyl” is a subset of alkyl and heteroaryl, as defined herein,and refers to an optionally substituted alkyl group substituted by anoptionally substituted heteroaryl group.

Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylgroups, as defined herein, which are divalent bridging groups arefurther referred to using the suffix -ene, e.g., alkylene, alkenylene,alkynylene, carbocyclylene, heterocyclylene, arylene, and heteroarylene.

As used herein, the term “optionally substituted” refers to asubstituted or unsubstituted moiety.

Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylgroups, as defined herein, are optionally substituted (e.g.,“substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted”alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or“unsubstituted” carbocyclyl, “substituted” or “unsubstituted”heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or“unsubstituted” heteroaryl group). In general, the term “substituted”,whether preceded by the term “optionally” or not, means that at leastone hydrogen present on a group (e.g., a carbon or nitrogen atom) isreplaced with a permissible substituent, e.g., a substituent which uponsubstitution results in a stable compound, e.g., a compound which doesnot spontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction. Unless otherwise indicated,a “substituted” group has a substituent at one or more substitutablepositions of the group, and when more than one position in any givenstructure is substituted, the substituent is either the same ordifferent at each position. The term “substituted” is contemplated toinclude substitution with all permissible substituents of organiccompounds, any of the substituents described herein that results in theformation of a stable compound. The present invention contemplates anyand all such combinations in order to arrive at a stable compound. Forpurposes of this invention, heteroatoms such as nitrogen may havehydrogen substituents and/or any suitable substituent as describedherein which satisfy the valencies of the heteroatoms and results in theformation of a stable moiety.

Exemplary carbon atom substituents include, but are not limited to,halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(aa), —ON(R^(bb))₂,—N(R^(bb))₂, —N(R^(bb))₃ ⁺X⁻, —N(OR^(cc))R^(bb), —SH, —SR^(aa),—SSR^(cc), —C(═O)R^(aa), —CO₂H, —CHO, —C(OR^(cc))₂, —CO₂R^(aa),—OC(═O)R^(aa), —OCO₂R^(aa), —C(═O)N(R^(bb))₂, —OC(═O)N(R^(bb))₂,—NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa), —NR^(bb)C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —OC(═NR^(bb))R^(aa),—OC(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂, —OC(═NR^(bb))N(R^(bb))₂,—NR^(bb)C(═NR^(bb))N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa),—NR^(bb)SO₂R^(aa), —SO₂N(R^(bb))₂, —SO₂R^(aa), —SO₂OR^(aa), —OSO₂R,—S(═O)R^(aa), —OS(═O)R^(aa), —Si(R^(aa))₃,—Osi(R^(aa))₃—C(═S)N(R^(bb))₂, —C(═O)SR^(aa), —C(═S)SR^(aa),—SC(═S)SR^(aa), —SC(═O)SR^(aa), —OC(═O)SR^(aa), —SC(═O)OR^(aa),—SC(═O)R^(aa), —P(═O)₂R^(aa), —OP(═O)₂R^(aa), —P(═O)(R^(aa))₂,—OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂,—OP(═O)₂N(R^(bb))₂, —P(═O)(NR^(bb))₂, —OP(═O)(NR^(bb))₂,—NR^(bb)P(═O)(OR^(cc))₂, —NR^(bb)P(═O)(NR^(bb))₂, —P(R^(cc))₂,—P(R^(cc))₃, —OP(R^(cc))₂, —OP(R^(cc))₃, —B(R^(aa))₂, —B(OR^(cc))₂,—BR^(aa)(OR^(cc)), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups; or two hydrogens ona carbon atom are replaced with the group ═O, ═S, ═NN(R^(bb))₂,═NNR^(bb)C(═O)R^(aa)═NNR^(bb)C(═O)OR^(aa), ═NNR^(bb)S(═O)₂R^(aa),═NR^(bb), or ═NOR^(cc); each instance of R^(aa) is, independently,selected from C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and5-14 membered heteroaryl, or two R^(aa) groups are joined to form a 3-14membered heterocyclyl or 5-14 membered heteroaryl ring, wherein eachalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylis independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;each instance of R^(bb) is, independently, selected from hydrogen, —OH,—OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂,—SO₂R^(cc), —SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc),—C(═S)SR^(cc), —P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂,—P(═O)(NR^(cc))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and5-14 membered heteroaryl, or two R^(bb) groups are joined to form a 3-14membered heterocyclyl or 5-14 membered heteroaryl ring, wherein eachalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylis independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;each instance of R^(cc) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups are joined to form a 3-14 memberedheterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl,alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups; eachinstance of R^(dd) is, independently, selected from halogen, —CN, —NO₂,—N₃, —SO₂H, —SO₃H, —OH, —OR^(ee), —ON(R^(ff))₂, —N(R^(ff))₂, —N(R^(ff))₃⁺X⁻, —N(OR^(ee))R^(ff), —SH, —SR^(ee), —SSR^(ee), —C(═O)R^(ee), —CO₂H,—CO₂R^(ee), —OC(═O)R^(ee), —OCO₂R^(ee), —C(═O)N(R^(ff))₂,—OC(═O)N(R^(ff))₂, —NR^(ff)C(═O)R^(ee), —NR^(ff)CO₂R^(ee),—NR^(ff)C(═O)N(R^(ff))₂, —C(═NR^(ff))OR^(ee), —OC(═NR^(ff))R^(ee),—OC(═NR^(ff))OR^(ee), —C(═NR^(ff))N(R^(ff))₂, —OC(═NR^(ff))N(R^(ff))₂,—NR^(ff)C(═NR^(ff))N(R^(ff))₂, —NR^(ff)SO₂R^(ee), —SO₂N(R^(ff))₂,—SO₂R^(ee), —SO₂OR^(ee), —OSO₂R^(ee), —S(═O)R^(ee), —Si(R^(ee))₃,—Osi(R^(ee))₃, —C(═S)N(R^(ff))₂, —C(═O)SR^(ee), —C(═S)SR^(ee),—SC(═S)SR^(ee), —P(═O)₂R^(ee), —P(═O)(R^(ee))₂, —OP(═O)(R^(ee))₂,—OP(═O)(OR^(ee))₂, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₁₀ carbocyclyl, 3-10 membered heterocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2, 3, 4, or 5 R^(gg) groups, or two R^(dd) substituents can be joinedto form ═O or ═S; each instance of R^(ee) is, independently, selectedfrom C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀carbocyclyl, C₆₋₁₀ aryl, 3-10 membered heterocyclyl, and 3-10 memberedheteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2, 3, 4, or 5 R^(gg) groups; each instance of R^(ff) is,independently, selected from hydrogen, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl, 3-10 memberedheterocyclyl, C₆₋₁₀ aryl and 5-10 membered heteroaryl, or two R^(ff)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2, 3, 4, or 5 R^(gg) groups; and each instance of R^(gg) is,independently, halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OC₁₋₆ alkyl,—ON(C₁₋₆ alkyl)₂, —N(C₁₋₆ alkyl)₂, —N(C₁₋₆ alkyl)₃ ⁺X⁻, —NH(C₁₋₆ alkyl)₂⁺X⁻, —NH₂(C₁₋₆ alkyl)⁺X⁻, —NH₃ ⁺X⁻, —N(OC₁₋₆ alkyl)(C₁₋₆ alkyl),—N(OH)(C₁₋₆ alkyl), —NH(OH), —SH, —SC₁₋₆ alkyl, —SS(C₁₋₆ alkyl),—C(═O)(C₁₋₆ alkyl), —CO₂H, —CO₂(C₁₋₆ alkyl), —OC(═O)(C₁₋₆ alkyl),—OCO₂(C₁₋₆ alkyl), —C(═O)NH₂, —C(═O)N(C₁₋₆ alkyl)₂, —OC(═O)NH(C₁₋₆alkyl), —NHC(═O)(C₁₋₆ alkyl), —N(C₁₋₆ alkyl)C(═O)(C₁₋₆ alkyl),—NHCO₂(C₁₋₆ alkyl), —NHC(═O)N(C₁₋₆ alkyl)₂, —NHC(═O)NH(C₁₋₆ alkyl),—NHC(═O)NH₂, —C(═NH)O(C₁₋₆ alkyl), —OC(═NH)(C₁₋₆ alkyl), —OC(═NH)OC₁₋₆alkyl, —C(═NH)N(C₁₋₆ alkyl)₂, —C(═NH)NH(C₁₋₆ alkyl), —C(═NH)NH₂,—OC(═NH)N(C₁₋₆ alkyl)₂, —OC(NH)NH(C₁₋₆ alkyl), —OC(NH)NH₂,—NHC(NH)N(C₁₋₆ alkyl)₂, —NHC(═NH)NH₂, —NHSO₂(C₁₋₆ alkyl), —SO₂N(C₁₋₆alkyl)₂, —SO₂NH(C₁₋₆ alkyl), —SO₂NH₂, —SO₂C₁₋₆ alkyl, —SO₂OC₁₋₆ alkyl,—OSO₂C₁₋₆ alkyl, —SOC₁₋₆ alkyl, —Si(C₁₋₆ alkyl)₃, —Osi(C₁₋₆alkyl)₃-C(═S)N(C₁₋₆ alkyl)₂, C(═S)NH(C₁₋₆ alkyl), C(═S)NH₂, —C(═O)S(C₁₋₆alkyl), —C(═S)SC₁₋₆ alkyl, —SC(═S)SC₁₋₆ alkyl, —P(═O)₂(C₁₋₆ alkyl),—P(═O)(C₁₋₆ alkyl)₂, —OP(═O)(C₁₋₆ alkyl)₂, —OP(═O)(OC₁₋₆ alkyl)₂, C₁₋₆alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl,C₆₋₁₀ aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl; or twoR^(gg) substituents can be joined to form ═O or ═S; wherein X⁻ is acounterion.

“Halo” or “halogen” refers to fluorine (fluoro, —F), chlorine (chloro,—Cl), bromine (bromo, —Br), or iodine (iodo, —I).

“Acyl” as used herein refers to a moiety selected from the groupconsisting of —C(═O)R^(aa), —CHO, —CO₂R^(aa), —C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂,—C(═O)NR^(bb)SO₂R^(aa), —C(═S)N(R^(bb))₂, —C(═O)SR^(aa), and—C(═S)SR^(aa), wherein R^(aa) and R^(bb) are as defined herein.

Nitrogen atoms can be substituted or unsubstituted as valency permits,and include primary, secondary, tertiary, and quaternary nitrogen atoms.Exemplary nitrogen atom substituents include, but are not limited to,hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),—C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(bb))R^(aa),—C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc),—SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc),—P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂, —P(═O)(NR^(cc))₂,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups attached to a nitrogen atom are joinedto form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl,and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5R^(dd) groups, and wherein R^(aa), R^(bb), R^(cc), and R^(dd) are asdefined above.

In certain embodiments, the substituent present on a nitrogen atom is anitrogen protecting group (also referred to as an amino protectinggroup). Nitrogen protecting groups include, but are not limited to, —OH,—OR^(aa), —N(R^(cc))₂, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))R^(aa), —C(═NR^(cc))OR^(aa),—C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),—SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc), C₁₋₁₀ alkyl(e.g., aralkyl, heteroaralkyl), C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aralkyl, aryl, and heteroaryl is independently substitutedwith 0, 1, 2, 3, 4, or 5 R^(dd) groups, and wherein R^(aa), R^(bb),R^(cc) and R^(dd) are as defined herein. Nitrogen protecting groups arewell known in the art and include those described in Protecting Groupsin Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^(rd) edition,John Wiley & Sons, 1999, incorporated herein by reference.

For example, nitrogen protecting groups such as amide groups (e.g.,—C(═O)R^(aa)) include, but are not limited to, formamide, acetamide,chloroacetamide, trichloroacetamide, trifluoroacetamide,phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

Nitrogen protecting groups such as carbamate groups (e.g.,—C(═O)OR^(aa)) include, but are not limited to, methyl carbamate, ethylcarbamate, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-Adamantyl)-1-methylethyl(Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzylcarbamate.

Nitrogen protecting groups such as sulfonamide groups (e.g.,—S(═O)₂R^(aa)) include, but are not limited to, p-toluenesulfonamide(Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide(Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to,phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacylderivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanylderivative, N-acetylmethionine derivative,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-hydroxyl, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate,N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In certain embodiments, the substituent present on an oxygen atom is anoxygen protecting group (also referred to as a hydroxyl protectinggroup). Oxygen protecting groups include, but are not limited to,—R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa),—C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂,—P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, whereinR^(aa), R^(bb), and R^(cc) are as defined herein. Oxygen protectinggroups are well known in the art and include those described inProtecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts,3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

Exemplary oxygen protecting groups include, but are not limited to,methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethyl silyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethyl silylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), methyl carbonate, 9-fluorenylmethylcarbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate(Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc),isobutyl carbonate, vinyl carbonate, allyl carbonate, t-butyl carbonate(BOC), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzylcarbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate,p-nitrobenzyl carbonate, S-benzyl thiocarbonate, 4-ethoxy-1-naphthylcarbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate,4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate,o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts).

In certain embodiments, the substituent present on a sulfur atom is asulfur protecting group (also referred to as a thiol protecting group).Sulfur protecting groups include, but are not limited to, —R^(aa),—N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa), —C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂,—S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃, —P(R^(cc))₂, —P(R^(cc))₃,—P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and—P(═O)(NR^(bb))₂, wherein R^(aa), R^(bb), and R^(cc) are as definedherein. Sulfur protecting groups are well known in the art and includethose described in Protecting Groups in Organic Synthesis, T. W. Greeneand P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporatedherein by reference.

As used herein, the term “leaving group” is given its ordinary meaningin the art of synthetic organic chemistry and refers to an atom or agroup capable of being displaced by a nucleophile. Examples of suitableleaving groups include, but are not limited to, halogen (such as F, Cl,Br, or I (iodine)), alkoxycarbonyloxy, aryloxycarbonyloxy,alkanesulfonyloxy, arenesulfonyloxy, alkyl-carbonyloxy (e.g., acetoxy),arylcarbonyloxy, aryloxy, methoxy, N,O-dimethylhydroxylamino, pixyl, andhaloformates. In some cases, the leaving group is a sulfonic acid ester,such as toluenesulfonate (tosylate, —OTs), methanesulfonate (mesylate,—OMs), p-bromobenzenesulfonyloxy (brosylate, —OBs), ortrifluoromethanesulfonate (triflate, —OTf). In some cases, the leavinggroup is a brosylate, such asp-bromobenzenesulfonyloxy. In some cases,the leaving group is a nosylate, such as 2-nitrobenzenesulfonyloxy. Insome embodiments, the leaving group is a sulfonate-containing group. Insome embodiments, the leaving group is a tosylate group. The leavinggroup may also be a phosphineoxide (e.g., formed during a Mitsunobureaction) or an internal leaving group such as an epoxide or cyclicsulfate. Other non-limiting examples of leaving groups are water,ammonia, alcohols, ether moieties, thioether moieties, zinc halides,magnesium moieties, diazonium salts, and copper moieties.

Exemplary α-GalCer analogs (GSLs with α-Glc) are used as immunologicadjuvants to accelerate, enhance, prolong, and/or modify or augment theeffects of a vaccine by stimulating the immune system of a patient whohas been vaccinated. In an exemplary implementation, the analog C34 isused as an adjuvant. As used herein, the term “alum adjuvant” refers toan aluminum salt with immune adjuvant activity, such as, for example,aluminum phosphate and aluminum hydroxide. These exemplary agents canadsorb and precipitates protein antigens in solution; the resultingprecipitate improves vaccine immunogenicity by facilitating the slowrelease of antigen from the vaccine depot formed at the site ofinoculation. Additionally, adjuvants contemplated herein can alsoinclude suitable organic adjuvants and suitable virosomes. In certainembodiments, exemplary organic adjuvants can include oil-based adjuvantssuch as squalene, MF59, QS-21 and AS03.

As used herein, the term “anti-tumor immunotherapy active agent” refersto antibody generated by a vaccine of the present disclosure thatinhibits, reduces and/or eliminates tumors, either alone and/or incombination with other synergistic agents.

Glycosphingolipids (GSLs) bearing α-galactosyl group (αGal) and phenylring on the acyl chain were known to be more potent than α-galactosylceramide (αGalCer) to stimulate both murine and human invariant NKT(iNKT) cells. Their activities in mice and humans correlated with thebinding avidities of the ternary interaction between iNKT TCR andCD1d-GSL complex.

The instant disclosure relates to the unexpected discovery that GSLswith glucose (αGlc) are stronger than those with αGal for humans butweaker for mice in the induction of cytokines/chemokines andexpansion/activation of immune cells. GSLs with glucose (αGlc) and Fderivatives of αGlc, and their impact on their immunostimulatoryactivities in humans are disclosed herein. The immune-stimulatorypotencies associated with the strength of ternary interaction for eachspecies are described herein. It is the iNKT TCR rather than CD1d thatdictates the species-specific responses, as demonstrated by mCD1d vs.hCD1d swapping assay disclosed herein. Glycosphingolipids (GSLs) withαGlc bear stronger ternary interaction and triggered more Th1-biasedimmunity as compared to GSLs with αGal in humans. GSLs with αGlc areless stimulatory than GSLs with αGal in mice. The species-specificresponses are attributed to the differential binding avidities ofternary complexes between species, reflecting the differences betweenmurine and human iNKT TCR as supported by mCD1d vs. hCD1d swapping assayas described herein.

These novel findings indicate differences in species and provide noveldesigns of GSL with modifications on the glycosyl group that are moreeffective for human therapy.

Compounds

The instant disclosure relates to exemplary immune adjuvant compounds ofFormula (I):

or pharmaceutically acceptable salt thereof; wherein R¹ is —OH orhalogen; R² is —OH, hydrogen or halogen; R³ is hydrogen or halogen; R⁴and R⁵ are each independently selected from the group consisting ofhydrogen, halogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted aryl, optionally substitutedheterocyclyl, optionally substituted heteroaryl, optionally substitutedalkoxy, an optionally substituted amino group, or optionally substitutedacyl; n is an integer of 1 to 15, inclusive; and m is an integer of 1 to20, inclusive.

In some embodiments of compound (I), R² is —OH. In some embodiments ofcompound (I), R² is hydrogen. In some embodiments of compound (I), R² ishalogen. In some embodiments of compound (I), R² is F. In someembodiments of compound (I), R² is Cl. In some embodiments of compound(I), R² is Br. In some embodiments of compound (I), R² is I.

In some embodiments of compound (I), R¹ is —OH. In some embodiments ofcompound (I), R¹ is halogen. In some embodiments of compound (I), R¹ isF. In some embodiments of compound (I), R¹ is Cl. In some embodiments ofcompound (I), R¹ is Br. In some embodiments of compound (I), R¹ is I.

In some embodiments of compound (I), R³ is hydrogen. In some embodimentsof compound (I), R³ is halogen. In some embodiments of compound (I), R³is F. In some embodiments of compound (I), R³ is Cl. In some embodimentsof compound (I), R³ is Br. In some embodiments of compound (I), R³ is I.

In some embodiments of compound (I), R¹ is —OH; R² is —OH, hydrogen orhalogen; and R³ is hydrogen or halogen. In some embodiments of compound(I), R¹ is —OH; R² is —OH; and R³ is hydrogen or halogen. In someembodiments of compound (I), R¹ is —OH; R² is hydrogen; and R³ ishydrogen or halogen. In some embodiments of compound (I), R¹ is —OH; R²is halogen; and R³ is hydrogen or halogen. In some embodiments ofcompound (I), R¹ is halogen; R² is —OH, hydrogen or halogen; and R³ ishydrogen or halogen. In some embodiments of compound (I), R¹ is halogen;R² is —OH; and R³ is hydrogen or halogen. In some embodiments ofcompound (I), R¹ is halogen; R² is hydrogen; and R³ is hydrogen orhalogen. In some embodiments of compound (I), R¹ is halogen; R² ishalogen; and R³ is hydrogen or halogen.

In some embodiments of compound (I), R⁴ is phenyl. In some embodiments,R⁴ is optionally substituted phenyl of Formula (II):

wherein i=0, 1, 2, 3, 4, or 5; each instance of R⁶ is independentlyselected from the group consisting of hydrogen, halogen, —CN, —NO₂, —N₃,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted phenyl, optionally substituted heterocyclyl, optionallysubstituted heteroaryl, optionally substituted alkoxy, an optionallysubstituted amino group, or optionally substituted acyl. In certainembodiments, i is 0. In certain embodiments, i is 1. In certainembodiments, i is 2. In certain embodiments, i is 3. In certainembodiments, i is 4. In certain embodiments, i is 5. In certainembodiments, i is 1 and R⁶ is halogen. In certain embodiments, i is 1and R⁴ is one of the formulae:

In certain embodiments, i is 2 and R⁴ is one of the formulae:

In certain embodiments, i is 3 and R⁴ is one of the formulae:

In certain embodiments, i is 4 and R⁴ is one of the formulae:

In certain embodiments, i is 5 and R⁴ is of the formula

In some embodiments of compound (I), R⁶ is halogen. In some embodimentsof compound (I), R⁶ is F. In some embodiments of compound (I), R⁶ is Cl.In some embodiments of compound (I), R⁶ is Br. In some embodiments ofcompound (I), R⁶ is I.

In some embodiments of compound (I), R⁴ is optionally substituted aryl.In some embodiments of compound (I), R⁴ is of Formula (III):

wherein j is 0, 1, 2, 3, or 4; k is 0, 1, 2, 3, 4, or 5; each instanceof R⁷ and R⁸ is independently selected from the group consisting ofhydrogen, halogen, —CN, —NO₂, —N₃, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted phenyl,optionally substituted heterocyclyl, optionally substituted heteroaryl,optionally substituted alkoxy, an optionally substituted amino group, oroptionally substituted acyl. In certain embodiments, k is 0. In certainembodiments, k is 1. In certain embodiments, k is 2. In certainembodiments, k is 3. In certain embodiments, k is 4. In certainembodiments, k is 5. In certain embodiments, k is 1 and R⁸ is halogen.In certain embodiments, k is 1 and R⁴ is one of the formulae:

In certain embodiments, k is 2 and R⁴ is one of the formulae:

In certain embodiments, k is 3 and R⁴ is one of the formulae:

In certain embodiments, k is 4 and R⁴ is one of the formulae:

In certain embodiments, k is 5 and R⁴ is of the formula

In some embodiments of compound (I), n is an integer of 1 to 15,inclusive. In some embodiments of compound (I), n is an integer of 5 to15, inclusive. In some embodiments of compound (I), n is an integer of10 to 15, inclusive. In some embodiments of compound (I), n is 10. Insome embodiments of compound (I), n is 11. In some embodiments ofcompound (I), n is 12. In some embodiments of compound (I), n is 13. Insome embodiments of compound (I), n is 14. In some embodiments ofcompound (I), n is 15.

In some embodiments of compound (I), m is an integer of 1 to 20,inclusive. In some embodiments of compound (I), m is an integer of 5 to20, inclusive. In some embodiments of compound (I), m is an integer of 5to 15, inclusive. In some embodiments of compound (I), m is an integerof 10 to 15, inclusive. In some embodiments of compound (I), m is 10. Insome embodiments of compound (I), m is 11. In some embodiments ofcompound (I), m is 12. In some embodiments of compound (I), m is 13. Insome embodiments of compound (I), m is 14. In some embodiments ofcompound (I), m is 15.

The In some embodiments of compound (I), R⁷ is hydrogen; R⁸ is F; and kis 1, 2 or 3. In some embodiments of compound (I), R⁷ is F; R⁸ is ahydrogen; and j is 1, 2 or 3. In some embodiments of compound (I), R⁷and R⁸ both are F; k is 1, 2 or 3; and j is 1, 2 or 3.

In some embodiments of formula (I), the provided compound is one of thefollowing compounds:

Pharmaceutical Compositions

The present disclosure provides pharmaceutical compositions comprisingan exemplary compound described herein and a pharmaceutically acceptableexcipient. The compositions disclosed herein can be included in apharmaceutical or nutraceutical composition together with additionalactive agents, carriers, vehicles, excipients, or auxiliary agentsidentifiable by a person skilled in the art upon reading of the presentdisclosure.

The pharmaceutical compositions preferably comprise at least onepharmaceutically acceptable carrier. In such pharmaceuticalcompositions, the compositions disclosed herein form the “activecompound,” also referred to as the “active agent.” As used herein thelanguage “pharmaceutically acceptable carrier” includes solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions. A pharmaceutical composition isformulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, e.g.,intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol, or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates, or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringes,or multiple dose vials made of glass or plastic.

Subject as used herein refers to humans and non-human primates (e.g.,guerilla, macaque, marmoset), livestock animals (e.g., sheep, cow,horse, donkey, and pig), companion animals (e.g., dog, cat), laboratorytest animals (e.g., mouse, rabbit, rat, guinea pig, hamster), captivewild animals (e.g., fox, deer), and any other organisms who can benefitfrom the agents of the present disclosure. There is no limitation on thetype of animal that could benefit from the presently described agents. Asubject regardless of whether it is a human or non-human organism may bereferred to as a patient, individual, animal, host, or recipient.

Pharmaceutical compositions suitable for an injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In allcases, the composition should be sterile and should be fluid to theextent that easy syringability exists. It should be stable under theconditions of manufacture and storage and be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Uses of Compositions Described Herein

The present invention provides compositions useful for stimulating animmune response in a human subject in need thereof, the methodcomprising: administering to the subject a therapeutically effectiveamount of a composition disclosed herein.

The compositions described herein can also be used to elevate invariantNatural Killer T (iNKT) cells production in a human subject in needthereof, the method comprising: administering to the subject in needthereof a therapeutically effective amount of a pharmaceuticallyacceptable composition, wherein the composition comprises an exemplarycompound disclosed herein.

The exemplary compositions described herein can also be used tostimulate cytokine and/or chemokine production in a human subject inneed thereof, the method comprising: administering to the subject atherapeutically effective amount of a pharmaceutically acceptablecomposition, wherein the composition comprises an amount sufficient toincrease cytokine/chemokine production, of a compound disclosed herein.

By an “effective” amount or a “therapeutically effective” amount of anactive agent is meant a nontoxic but sufficient amount of the agent toprovide a beneficial effect. The amount of active agent that is“effective” will vary from subject to subject, depending on the age andgeneral condition of the individual, the particular active agent oragents, and the like. Unless otherwise indicated, the term“therapeutically effective” amount as used herein is intended toencompass an amount effective for the prevention of an adverse conditionand/or the amelioration of an adverse condition, i.e., in addition to anamount effective for the treatment of an adverse condition.

As defined herein, a therapeutically effective amount of the activecompound (i.e., an effective dosage) may range from about 0.001 to 100g/kg body weight, or other ranges that would be apparent and understoodby artisans without undue experimentation. The skilled artisan willappreciate that certain factors can influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health or age of the subject, and other diseases present.

An adverse condition as that term is used herein may be a “normal”condition that is frequently seen in individuals or a pathologiccondition that may or may not be associated with a named disease.

As used herein, the term “lipid” refers to any fat-soluble (lipophilic)molecule that participates in cell signaling pathways.

As used herein, the term “glycolipid” refers to a carbohydrate-attachedlipid that serves as a marker for cellular recognition.

According to another aspect, one or more kits of parts can be envisionedby the person skilled in the art, the kits of parts to perform at leastone of the methods herein disclosed, the kit of parts comprising two ormore compositions, the compositions comprising alone or in combinationan effective amount of the compositions disclosed herein according tothe at least one of the above mentioned methods.

The kits possibly include also compositions comprising active agents,identifiers of a biological event, or other compounds identifiable by aperson skilled upon reading of the present disclosure. The kit can alsocomprise at least one composition comprising an effective amount of thecompositions disclosed herein or a cell line. The compositions and thecell line of the kits of parts to be used to perform the at least onemethod herein disclosed according to procedure identifiable by a personskilled in the art.

As used herein, the term “specifically binding,” refers to theinteraction between binding pairs (e.g., an antibody and an antigen). Invarious instances, specifically binding can be embodied by an affinityconstant of about 10⁻⁶ moles/liter, about 10⁻⁷ moles/liter, or about10⁻⁸ moles/liter, or less.

As will be apparent to those of skill in the art upon reading thisinvention, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

In one aspect, the immune composition described herein can beadministered parenterally (e.g., intravenous injection, subcutaneousinjection or intramuscular injection). Alternatively, other modes ofadministration including suppositories and oral formulations may bedesirable. For suppositories, binders and carriers may include, forexample, polyalkalene glycols or triglycerides. Oral formulations mayinclude normally employed incipients such as, for example,pharmaceutical grades of saccharine, cellulose, magnesium carbonate andthe like. These compositions take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations or powders andcontain 10-95% of the immune composition described herein.

The immune composition is administered in a manner compatible with thedosage formulation, and in an amount that is therapeutically effective,protective and immunogenic. The quantity to be administered depends onthe subject to be treated, including, for example, the capacity of theindividual's immune system to synthesize antibodies, and if needed, toproduce a cell-mediated immune response. Precise amounts of activeingredient required to be administered depend on the judgment of thepractitioner. However, suitable dosage ranges are readily determinableby one skilled in the art. Suitable regimes for initial administrationand booster doses are also variable, but may include an initialadministration followed by subsequent administrations. The dosage of thevaccine may also depend on the route of administration and variesaccording to the size of the host.

The immune composition of this invention can also be used to generateantibodies in animals for production of antibodies, which can be used inboth cancer treatment and diagnosis. Methods of making monoclonal andpolyclonal antibodies and fragments thereof in animals (e.g., mouse,rabbit, goat, sheep, or horse) are well known in the art. See, forexample, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, New York. The term “antibody” includes intactimmunoglobulin molecules as well as fragments thereof, such as Fab,F(ab′)₂, Fv, scFv (single chain antibody), and dAb (domain antibody;Ward, et. al. (1989) Nature, 341, 544).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In the case of conflict, thepresent document, including definitions will control. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention, thepreferred methods and materials are now described. All publications andpatents specifically mentioned herein are incorporated by reference forall purposes including describing and disclosing the chemicals, celllines, vectors, animals, instruments, statistical analysis andmethodologies which are reported in the publications which might be usedin connection with the invention. All references cited in thisspecification are to be taken as indicative of the level of skill in theart. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

Before the present materials and methods are described, it is understoodthat this invention is not limited to the particular methodology,protocols, materials, and reagents described, as these may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included

Examples

The following examples are put forth so as to provide those skilled inthe art with a complete invention and description of how to make and useembodiments in accordance with the invention, and are not intended tolimit the scope of what the inventors regard as their discovery. Effortshave been made to ensure accuracy with respect to numbers used (e.g.amounts, temperature, etc.) but some experimental errors and deviationsshould be accounted for. Unless indicated otherwise, parts are parts byweight, molecular weight is weight average molecular weight, temperatureis in degrees Centigrade, and pressure is at or near atmospheric.

General:

All reagent chemicals were purchased as reagent grade and used withoutfurther purification. Anhydrous solvents such as dichloromethane(CH₂Cl₂), tetrahydrofuran (THF), N,N-dimethylformamide (DMF), methanol(MeOH), pyridine were purchased from Acros. HPLC grade solventschloroform (CHCl₃) and methanol were purchased from Merck. Molecularsieves 4 Å (MS 4 Å) for glycosylation was purchased from Acros andactivated by flame. Reactions were monitored with analytical thin layerchromatography (TLC) in EM silica gel 60 F254 plates and visualizedunder UV (254 nm) and/or staining with acidic ceric ammonium molybdateor ninhydrin. Flash column chromatography was performed on silica gel 60Geduran (40-63 μm, Merck). Biogel LH20 for purification of finalproducts was purchased from Aldrich. ¹H NMR spectra were recorded on aBruker Topspin-600 (600 MHz) spectrometer at 20° C. Chemical shifts (δppm) were assigned according to the internal standard signal of CDCl₃(δ=7.24 ppm), MeOD (δ=3.31 ppm), and pyridine-d⁵ (δ=7.58 ppm). ¹³C NMRspectra were obtained on a Bruker Topspin-600 (150 MHz) spectrometer andwere reported in δ ppm scale using the signal of CDCl₃ (δ=77.23 ppm),MeOD (δ=49.15 ppm) for calibration. Coupling constants (J) are reportedin Hz. Splitting patterns are described by using the followingabbreviations: s, singlet; d, doublet; t, triplet; dd, double doublet;m, multiplet. ¹H NMR spectra are reported in this order: chemical shift;multiplicity; number(s) of proton; coupling constant(s).

Chemical Syntheses Synthesis of Glucosyl Donor 8

Compound 2:

To a solution of 1,2,3,4,6-Penta-O-acetyl-3-D-glucopyranose 1 (40 g,102.5 mmol) in 200 mL of dry CH₂Cl₂ was added p-toluenethiol (15.4 g,123 mmol) and BF₃OEt₂ (15.4 mL, 123 mmol) at 0° C., the reaction wasstirred for 16 h at ambient temperature under argon. The resultingsolution was directly extracted with saturated NaHCO₃ solution andbrine, dried over MgSO₄ and evaporated. Followed by recrystallization ina solution of AcOEt-hexanes to give 2 as white solid (32.6 g, 70%). ¹HNMR (CDCl₃, 600 MHz) δ 7.36 (2H, d, J=7.2 Hz), 7.10 (2H, d, J=7.8 Hz),5.18 (1H, t, J=9.0 Hz), 5.00 (1H, t, J=9.6 Hz), 4.91 (1H, t, J=9.0 Hz),4.61 (1H, d, J=10.2 Hz), 4.14-4.20 (2H, m), 3.67 (1H, s), 2.33 (3H, s),2.07 (3H, s), 2.06 (3H, s,), 1.99 (3H, s), 1.96 (3H, s). ¹³C NMR (CDCl₃,150 MHz) δ 170.78, 170.40, 169.59, 169.45, 139.00, 134.04, 129.88,127.73, 86.03, 75.94, 74.21, 70.10, 68.38, 62.32, 21.39, 20.97, 20.94,20.79, 20.78. HRMS (ESI-TOF) for C₂₁H₂₆O₉SNa⁺ [M+Na]⁺ calcd 477.1190,found 477.1201.

Compound 3:

To a solution of 2 (32.6 g, 71.8 mmol) in 500 mL of dry MeOH was addedcatalytic amount of sodium methoxide (NaOMe) and stirred for 3 h atambient temperature. The reaction was neutralized by adding AmberliteIR-120 and filtered, the resulting solution was concentrated to drynessto give 3 (20.3 g, 99%) as white solid, which was directly used for nextreaction without further purification. ¹H NMR (MeOD, 600 MHz) δ 7.46(2H, d, J=7.8 Hz), 7.12 (2H, d, J=7.8 Hz), 4.50 (1H, d, J=9.6 Hz), 3.85(1H, d, J=12.6, 1.8 Hz), 3.66 (1H, dd, J=12.0, 5.4 Hz), 3.36 (1H, t,J=9.0 Hz), 3.24-3.28 (2H, m), 3.17 (1H, t, J=9.0 Hz), 2.13 (3H, s). ¹³CNMR (MeOD, 150 MHz) δ 138.90, 133.66, 131.33, 130.67, 89.79, 82.16,79.81, 73.82, 71.50, 63.03, 21.24. HRMS (ESI-TOF) for C₁₃H8O₅SNa⁺[M+Na]⁺ calcd 309.0767, found 309.0772.

Compound 4:

To a solution of 3 (11.1 g, 38.8 mmol) in 48 mL of dry pyridine wasadded triphenylmethyl chloride (13.5 g, 46.6 mmol). The reaction wasstirred for 16 h at 60° C. under argon. After removal of the solvent,the mixture was purified by flash column chromatography on silica gel(hexanes:AcOEt:MeOH 1:1:0.1) to give 4 (12.3 g, 60%) as white powder. ¹HNMR (MeOD, 600 MHz) δ 7.56 (2H, d, J=7.8 Hz), 7.47 (6H, d, J=7.8 Hz),7.27 (6H, t, J=7.2 Hz), 7.22 (3H, t, J=7.2 Hz), 7.05 (2H, d, J=8.4 Hz),4.58 (1H, d, J=9.6 Hz), 3.40-3.43 (2H, m), 3.31 (1H, m), 3.23-3.27 (3H,m), 2.27 (3H, s). ¹³C NMR (MeOD, 150 MHz) δ 145.69, 138.71, 133.62,131.51, 130.77, 130.16, 128.87, 128.13, 89.46, 87.88, 80.98, 80.02,73.93, 71.85, 65.14, 21.34. HRMS (ESI-TOF) for C₃₂H₃₂O₅SNa⁺ [M+Na]⁺calcd 551.1863, found 551.1876.

Compound 5:

To a solution of 4 (21.1 g, 39.9 mmol) in 200 mL of dryN,N-dimethylformamide (DMF) was added sodium hydride (60% in mineraloil) (5.8 g, 143.6 mmol) at 0° C. The reaction was stirred for 1 h,followed by the addition of benzyl bromide (17.2 mL, 143.6 mmol) thenstirred for 16 h under argon at ambient temperature. The reaction wasquenched by MeOH and evaporated to dryness. The residue was diluted withAcOEt, the solution was washed with H₂O and brine, dried over MgSO₄, andevaporated to dryness. The mixture was purified by flash columnchromatography on silica gel (hexanes:AcOEt 9:1) to give 5 (22.3 g, 70%)as white powder. ¹H NMR (CDCl₃, 600 MHz) δ 7.60 (2H, d, J=7.8 Hz), 7.50(6H, d, J=7.8 Hz), 7.42 (2H, d, J=7.2 Hz), 7.34 (2H, t, J=7.2 Hz),7.24-7.32 (12H, m), 7.18-7.23 (4H, m), 7.14-7.17 (2H, m), 7.05 (2H, d,J=7.8 Hz), 6.82 (2H, d, J=7.8 Hz), 4.91 (1H, d, J=10.8 Hz), 4.84 (1H, d,J=10.8 Hz), 4.80 (1H, d, J=10.8 Hz), 4.74 (1H, d, J=10.2 Hz), 4.64 (2H,dd, J=9.6, 5.4 Hz), 4.30 (1H, d, J=10.2 Hz), 3.74 (1H, t, J=9.6 Hz),3.64 (1H, t, J=8.4 Hz), 3.60 (1H, d, J=9.6 Hz), 3.55 (1H, t, J=9.6 Hz),3.42 (1H, m), 3.25 (1H, dd, J=10.2, 4.2 Hz), 2.30 (3H, s). ¹³C NMR(CDCl₃, 150 MHz) δ 144.14, 138.57, 138.43, 137.94, 137.90, 133.01,130.10, 129.96, 129.07, 128.73, 128.67, 128.45, 128.42, 128.31, 128.20,128.16, 128.15, 128.07, 128.01, 127.89, 127.20, 87.90, 87.07, 86.70,80.96, 79.04, 78.04, 76.25, 75.61, 75.21, 62.68, 45.18, 21.37. HRMS(ESI-TOF) for C₅₃H₅₀O₅SNa⁺ [M+Na]⁺ calcd 821.3271, found 821.3310

Compound 6:

To a solution of 5 (30.0 g, 37.5 mmol) in 1065 mL of aqueous acetic acidsolution (AcOH:H₂O 4:1) was stirred for 3 h at 75° C. After removal ofthe solvent, the residue was purified by flash column chromatography onsilica gel (hexanes:AcOEt 2:1) to give 6 (16.7 g, 80%) as white solid.¹H NMR (CDCl₃, 600 MHz) δ 7.37-7.41 (4H, m), 7.25-7.33 (13H, m), 7.10(2H, d, J=7.8 Hz), 4.91 (1H, d, J=10.2 Hz), 4.89 (1H, d, J=10.8 Hz),4.84 (1H, d, J=10.8 Hz), 4.83 (1H, d, J=10.8 Hz), 4.74 (1H, d, J=10.8Hz), 4.62 (1H, d, J=10.2 Hz), 3.83-3.86 (1H, m), 3.65-3.71 (2H, m), 3.54(1H, t, J=9.6 Hz), 3.44 (1H, t, J=9.0 Hz), 3.33-3.36 (1H, m), 2.31 (3H,s), 1.87 (1H, t, J=6.6 Hz). ¹³C NMR (CDCl₃, 150 MHz) δ 138.51, 138.24,138.14, 138.02, 132.85, 129.99, 129.63, 128.71, 128.66, 128.63, 128.40,128.22, 128.16, 128.09, 127.98, 127.94, 87.99, 86.75, 81.27, 79.43,77.81, 76.01, 75.69, 75.30, 62.34, 21.31. HRMS (ESI-TOF) forC₃₄H₃₆O₅SNa⁺ [M+Na]⁺ calcd 579.2176, found 579.2188.

Compound 7:

To a solution of 6 (5.0 g, 9.0 mmol) in 18 mL of dry pyridine was addedacetic anhydride (1.0 mL). The reaction was stirred for 16 h at ambienttemperature under argon. After removal of the solvent, the residue wasdiluted with AcOEt, the solution was washed with H₂O and brine, driedover MgSO₄ and evaporated to dryness. The mixture was purified by flashcolumn chromatography on silica gel (hexanes:AcOEt 5:1) to give 7 (5.3g, 99%) as white solid. ¹H NMR (CDCl₃, 600 MHz) δ 7.44 (2H, d, J=7.8Hz), 7.38 (2H, d, J=7.8 Hz), 7.26-7.34 (11H, m), 7.22-7.24 (2H, m), 7.08(2H, d, J=7.8 Hz), 4.91 (1H, d, J=10.2 Hz), 4.90 (1H, d, J=11.4 Hz),4.83 (1H, d, J=10.8 Hz), 4.82 (1H, d, J=10.8 Hz), 4.71 (1H, d, J=10.2Hz), 4.57 (1H, d, J=9.6 Hz), 4.55 (1H, d, J=10.8 Hz), 4.34 (1H, d,J=12.0 Hz), 4.17-4.20 (1H, m), 3.67-3.70 (1H, m), 3.49-3.50 (2H, m),3.45 (1H, t, J=9.6 Hz), 2.32 (3H, s), 2.03 (3H, s). ¹³C NMR (CDCl₃, 150MHz) δ 170.90, 138.44, 138.16, 138.13, 137.81, 132.98, 129.85, 128.76,128.72, 128.68, 128.45, 128.30, 128.26, 128.15, 128.02, 88.00, 86.93,81.07, 77.74, 76.08, 75.68, 75.32, 63.51, 21.35, 21.09. HRMS (ESI-TOF)for C₃₆H₃₈O₆SNa⁺ [M+Na]⁺ calcd 621.2281, found 621.2301.

Compound 8:

To a solution of 7 (5.5 g, 9.2 mmol) in 129 mL of aqueous acetonesolution (acetone:H₂O 4:1) was added N-bromosuccinimide (1.7 g, 9.5mmol). The reaction was stirred for 1 h at ambient temperature. Afterremoval of the solvent, the residue was diluted with AcOEt, extractedwith H₂O, aqueous sodium thiosulfate (NaS₂O₃) solution, brine then driedover MgSO₄. The mixture was purified by flash column chromatography onsilica gel (hexane:AcOEt 2:1) to give 8 (3.1 g, 69%, α/β=1:1) as whitesolid. ¹H NMR (CDCl₃, 600 MHz) δ 7.24-7.35 (30H, m), 5.18 (1H, t, J=3.0Hz), 4.96 (1H, d, J=10.2 Hz), 4.94 (2H, d, J=10.8 Hz), 4.86 (1H, d,J=10.8 Hz), 4.85 (1H, d, J=10.2 Hz), 4.84 (1H, d, J=10.2 Hz), 4.80 (1H,d, J=10.8 Hz), 4.76 (2H, d, J=11.4 Hz), 4.71 (1H, dd, J=7.2, 5.4 Hz),4.68 (1H, d, J=12.0 Hz), 4.55 (2H, d, J=10.8 Hz), 4.34 (1H, dd, J=12.0,1.2 Hz), 4.23-4.28 (2H, m), 4.17 (1H, dd, J=12.0, 4.8 Hz), 4.06-4.09(1H, m), 3.98 (1H, t, J=9.6 Hz), 3.67 (1H, t, J=8.4 Hz), 3.50-3.56 (3H,m), 3.48 (1H, t, J=9.0 Hz), 3.37-3.40 (2H, m), 3.01 (1H, d, J=3.0 Hz),2.02 (3H, s), 2.01 (3H, s). ¹³C NMR (CDCl₃, 150 MHz) δ 138.64, 138.49,138.39, 137.96, 137.89, 137.81, 128.75, 128.70, 128.68, 128.65, 128.63,128.33, 128.29, 128.27, 128.22, 128.20, 128.14, 128.06, 127.99, 127.93,97.62, 91.33, 84.71, 83.20, 81.82, 80.18, 77.39, 75.96, 75.92, 75.23,75.21, 74.98, 73.47, 73.19, 69.02, 63.35, 63.27, 21.06. HRMS (ESI-TOF)for C₂₉H₃₂O₇Na⁺ [M+Na]⁺ calcd 515.2040, found 515.2052.

Synthesis of Acceptor 18

Compound 13:

To a solution of D-Lyxose 12 (20 g, 133 mmol) in 200 mL of anhydrousN,N-dimethylformamide (DMF) was added 2-methoxypropene (15 mL, 160 mmol)and camphor-10-sulfonic acid (CSA) (3 g, 13.3 mmol) at 0° C. Thereaction was stirred for 16 h at ambient temperature under argon. Thesolution was quenched with triethylamine (Et₃N), evaporated to drynessand directly purified by flash column chromatography on silica gel(hexanes:AcOEt:MeOH 1:1:0.2) to give 13 (21 g, 83%) as white solid. ¹HNMR (CDCl₃, 600 MHz): δ 5.22 (s, 1H), 4.78 (dd, 1H, J=6.0, 3.6 Hz), 4.53(d, 1H, J=6.0 Hz), 4.17 (m, 1H, J=6.6, 4.8 Hz), 3.82 (dd, 1H, J=11.7,4.8 Hz), 3.71 (dd, 1H, J=11.7, 6.6 Hz), 1.40 (s, 3H), 1.29 (s, 3H). ¹³CNMR (CDCl₃, 150 MHz): δ 113.62, 102.27, 87.42, 81.73, 81.35, 61.37,26.46, 25.02. HRMS (ESI-TOF) for C₈H₁₄O₅Na⁺ [M+Na]⁺ calcd 213.0733,found 213.0751.

Compound 14:

To a stirred solution of 13 (21 g, 110 mmol) in 140 mL of dry pyridinewas added triphenylmethyl chloride (37.8 g, 132 mmol). The reaction wasstirred for 16 h at 60° C. under argon. The solution was concentrated todryness, the residue was dissolved with ethyl acetate (AcOEt), washedwith H₂O, brine and dried over magnesium sulfate (MgSO₄) thenevaporated. The mixture was purified by flash column chromatography onsilica gel (hexanes:AcOEt 1:2) to give 14 (36.5 g, 77%) as white powder.¹H NMR (CDCl₃, 600 MHz): δ 7.46 (m, 6H), 7.27 (m, 6H), 7.21 (m, 3H),5.36 (d, 1H, J=1.8 Hz), 4.73 (dd, 1H, J=6.0, 4.8 Hz), 4.57 (d, 1H, J=6.0Hz), 4.31 (ddd, 1H, J=4.8, 4.8, 7.8 Hz), 3.41 (dd, 1H, J=9.6, 4.8 Hz),3.37 (dd, 1H, J=9.6, 7.8 Hz), 2.41 (m, 1H, J=1.8 Hz), 1.27 (s, 3H), 1.25(s, 3H). ¹³C NMR (CDCl₃, 150 MHz): δ 143.95, 128.82, 127.75, 126.95,112.48, 101.22, 86.85, 85.41, 80.11, 79.70, 61.85, 26.02, 25.09. HRMS(ESI-TOF) for C₂₇H₂₈O₅Na⁺ [M+Na]⁺ calcd 455.1829, found 455.1833.

Compound 15:

To a stirred solution of 14 (8.4 g, 19.4 mmol) in 40 mL of anhydroustetrahydrofurane (THF) was added lithium bis(trimethylsilyl)amide(LHMDS) (20 mL of 1M solution in THF, 20 mmol) at 0° C., the reactionwas stirred for 1 h under argon. To a stirred solution of Wittig reagentC₁₃H₂₇PPh₃Br (20.1 g, 38.2 mmol), prepared from 1-bromotridecane(C₁₃H₂₇Br) and triphenylphosphine (PPh₃) refluxed in toluene for 5 days,in 83 mL of anhydrous THF was added LHMDS (40 mL of 1M solution in THF,40 mmol) at 0° C., the reaction was stirred for 1 h under argon toproduce the bright orange ylide. The solution of 14 was added dropwiseto the ylide at 0° C., and the reaction was allowed to warm to ambienttemperature and stirred for 9 h under argon. The resulting solution wasquenched with MeOH and evaporated to dryness. The residue was dilutedwith AcOEt, extracted with H₂O and brine, dried over MgSO₄ thenconcentrated. The mixture was purified by flash column chromatography onsilica gel (hexanes:AcOEt 15:1) to give 15 (8.7 g, 75%) as colorlessoil. ¹H NMR (CDCl₃, 600 MHz): δ 7.40-7.45 (m, 9H), 7.25-7.30 (m, 9H),7.19-7.23 (m, 5H), 5.49-5.57 (m, 3H, J=6.6 Hz), 4.90 (t, 1H, J=6.6 Hz),4.43 (t, 1.5H, J=6.6 Hz), 4.25 (dd, 0.5H, J=6.6, 4.6 Hz), 4.20 (dd, 1H,J=6.6, 4.5 Hz), 3.74 (m, 1H), 3.68 (m, 0.5H), 3.22 (dd, 0.5H, J=9.6, 5.0Hz), 3.15 (dd, 1H, J=9.3, 5.1 Hz), 3.10 (m, 1.5H), 2.37 (m, 1.5H),1.90-2.00 (m, 2H), 1.75 (m, 1H), 1.47 (m, 5H), 1.37 (m, 5H), 1.19-1.33(m, 35H), 0.86 (t, 5H, J=7.1 Hz). ¹³C NMR (CDCl₃, 150 MHz): δ 144.07,137.58, 135.56, 128.90, 128.02, 127.24, 125.41, 125.15, 108.58, 108.50,86.90, 86.84, 79.14, 77.86, 77.74, 73.21, 69.51, 69.43, 65.19, 64.84,32.45, 32.13, 29.89, 29.86, 29.81, 29.71, 29.69, 29.57, 29.50, 29.47,29.11, 27.80, 27.59, 27.55, 25.26, 22.91, 14.35. HRMS (ESI-TOF) forC₄₀H₅₄O₄Na⁺ [M+Na]⁺ calcd 621.3914, found 621.3919.

Compound 16:

Compound 16 was prepared from catalytic hydrogenation of 15 (1 g, 1.7mmol) in 10 mL of anhydrous MeOH containing catalytic amount ofpalladium hydroxide on carbon (20% Pd). The suspension was stirred for 4h in an H₂ atmosphere. The solution was filtered through Celite 545 toremove the catalyst, evaporated to dryness then purified by flash columnchromatography on silica gel (hexane:AcOEt 20:1) to give 16 (903 mg,90%) as white solid. ¹H NMR (CDCl₃, 600 MHz): δ 7.43 (m, 6H), 7.27 (m,6H), 7.21 (m, 3H), 4.12 (dd, 1H, J=6.4, 3.7 Hz), 4.05 (ddd, 1H, J=9.9,6.4, 3.6 Hz), 3.69 (m, 1H, J=6.0, 6.0, 5.8, 3.7 Hz), 3.18 (m, 2H, J=9.5,9.5, 6.0, 5.8 Hz), 2.29 (d, 1H, J=6.0 Hz), 1.60-1.67 (m, 1H), 1.45-1.49(m, 1H), 1.43 (s, 3H), 1.34-1.38 (m, 1H), 1.33 (s, 3H), 1.20-1.30 (m,23H), 0.86 (t, 3H, J=7.2 Hz). ¹³C NMR (CDCl₃, 150 MHz): δ 144.09,128.91, 128.05, 127.26, 107.95, 87.02, 77.67, 69.15, 65.43, 32.15,29.92, 29.88, 29.83, 29.77, 29.58, 27.60, 26.99, 25.43, 22.92, 14.36.HRMS (ESI-TOF) for C₄₀H₅₆O₄Na⁺ [M+Na]⁺ calcd 623.4071, found 623.4112.

Compound 17:

To a solution of 16 (5 g, 8.3 mmol) and 4 Å molecular sieves (1 g) in 39mL of anhydrous CH₂Cl₂ was added 2,6-lutidine (3.5 mL, 30 mmol) atambient temperature. When the solution was cooled to −45° C.,trifluoromethanesulfonic anhydride (Tf₂O) (2.67 mL, 15.9 mmol) was addeddropwise and the reaction was stirred for 1 h under argon. Followed bythe addition of tetramethylguanidinium azide (TMGA) (3.9 g, 25 mmol),the reaction was allowed to warm to ambient temperature and stirred for16 h under argon. The resulting solution was filtered through Celite 545to remove 4 Å molecular sieves and the residue was diluted with CH₂Cl₂,extracted with H₂O and brine, dried over MgSO₄ then evaporated. Themixture was simply purified by flash column chromatography on silica gel(hexanes:AcOEt 20:1) to remove most of the impurities and directly usedfor the next step.

Compound 18:

To a solution of 17 in 20 mL of anhydrous CH₂Cl₂ was addedtetrafluoroacetic acid/tetrafluoroacetic anhydride (TFA/TFAA 1.8 M/1.8 Min CH₂Cl₂) (14 mL, 24.9 mmol) at 4° C. and stirred for 15 min underargon. The reaction was quenched by adding 10 mL of Et₃N then pouredinto 200 mL of methanol (MeOH) and stirred for another 15 min. Afterremoval of the solvent, the residue was diluted with AcOEt, extractedwith H₂O, saturated NaHCO₃ solution and brine then dried over MgSO₄. Theorganic layer was concentrated in vacuo and the mixture was purified byflash column chromatography on silica gel (hexanes:AcOEt 10:1) to give18 (2 g, two steps 63%) as yellow oil. ¹H NMR (CDCl₃, 600 MHz): δ 4.16(ddd, 1H, J=9.7, 5.6, 3.6 Hz), 3.97 (dd, 1H, J=11.6, 4.2 Hz), 3.94 (dd,1H, J=9.4, 5.6 Hz), 3.85 (dd, 1H, J=11.6, 5.4 Hz), 3.45 (ddd, 1H, J=9.4,5.4, 4.2 Hz), 1.50-1.62 (m, 2H, J=9.7, 3.6 Hz), 1.41 (s, 3H), 1.31-1.37(m, 6H), 1.22-1.30 (m, 22H), 0.86 (t, 3H, J=6.9, 6.9 Hz). ¹³C NMR(CDCl₃, 150 MHz): δ 108.66, 77.96, 76.91, 64.18, 61.39, 32.14, 29.90,29.87, 29.81, 29.80, 29.75, 29.60, 29.58, 28.25, 26.74, 25.77, 22.91,14.34. HRMS (ESI-TOF) for C₂₁H₄₁N₃O₃H⁺ [M+H]⁺ calcd 383.3148, found356.3157 (—N₂).

Synthesis of α-Glucosylceramide Analogues 24-26

Compound 19:

To a solution of glucosyl donor 8 (2.9 g, 5.9 mmol), dimethylsulfide(590 μL, 7.8 mmol), 4 Å molecular sieve (500 mg) and 2-chloropyridine(1.8 mL, 19.5 mmol) in anhydrous CH₂Cl₂ (15 mL) was addedtrifluoromethanesulfonic anhydride (1 mL, 6 mmol) at −45° C. underargon. The reaction was stirred for 20 min at −45° C., 20 min at 0° C.and another 20 min at ambient temperature, followed by the addition ofacceptor 18 (1.5 g, 3.9 mmol) in 5 mL of CH₂Cl₂. The reaction wasstirred for 16 h at ambient temperature under argon. The solution wasfiltered through Celite 545 to remove molecular sieve. After removal ofthe solvent, the residue was diluted with AcOEt, the solution was washedwith H₂O and brine, dried over MgSO₄ and evaporated to dryness. Themixture was purified by flash column chromatography on silica gel(hexanes:AcOEt 10:1) to give 19 as colorless oil (2 g, 60%). ¹H NMR(CDCl₃, 600 MHz) δ 7.36 (2H, d, J=7.8 Hz), 7.24-7.33 (13H, m), 4.97 (1H,d, J=10.8 Hz), 4.86 (1H, d, J=10.8 Hz), 4.85 (1H, d, J=3.6 Hz), 4.78(1H, d, J=10.8 Hz), 4.72 (1H, d, J=12.0 Hz), 4.68 (1H, d, J=12.0 Hz),4.54 (1H, d, J=10.8 Hz), 4.21-4.26 (2H, m), 4.08-4.11 (1H, m), 4.02-4.07(2H, m), 3.97 (1H, dd, J=9.6, 5.4 Hz), 3.84-3.87 (1H, m), 3.60 (1H, dd,J=10.8, 7.2 Hz), 3.54 (1H, dd, J=9.6, 3.6 Hz), 3.44-3.48 (2H, m), 2.00(3H, s), 1.57-1.61 (1H, m), 1.50-1.55 (1H, m), 1.37 (3H, s), 1.22-1.35(27H, m), 0.86 (3H, t, J=7.2 Hz). ¹³C NMR (CDCl₃, 150 MHz) δ 170.93,138.82, 138.58, 138.05, 128.68, 128.61, 128.59, 128.34, 128.31, 128.13,127.90, 127.86, 81.82, 80.26, 77.97, 77.30, 75.93, 75.61, 75.24, 72.92,69.50, 69.34, 63.26, 59.99, 32.13, 29.90, 29.87, 29.82, 29.77, 29.57,29.54, 28.42, 26.73, 25.89, 22.90, 21.03, 14.33. HRMS (ESI-TOF) forC₅₀H₇₁N₃O₉Na⁺ [M+Na]⁺ calcd 880.5083, found 880.5124.

Compound 20:

To a solution of 19 (269 mg, 0.31 mmol) in pyridine/H₂O (10:1, 12 mL)was added triphenylphosphine (165 mg, 0.63 mmol). The reaction wasstirred for 16 h at 45° C. under argon. After removal of the solvent,the residue was diluted with AcOEt, extracted with H₂O, brine and driedover MgSO₄ then evaporated to dryness. The mixture was used for nextstep without prior purification.

Compound 21:

To a solution of compound 20 in 36 mL of anhydrous CH₂Cl₂ was addedhexacosanoic acid (159 mg, 0.4 mmol), Et₃N (88 μL), EDC (90 mg, 0.47mmol) and HBTu (178 mg, 0.47 mmol). The reaction was stirred for 16 h atambient temperature under argon. After removal of the solvent, theresidue was diluted with AcOEt, extracted with H₂O, brine and dried overMgSO₄ then evaporated to dryness. The mixture was purified by flashcolumn chromatography on silica gel (hexanes:AcOEt 4:1) to give 21 aswhite solid (293 mg, 78%, two steps).

Compound 22:

To a solution of 21 (293 mg, 0.24 mmol) in 50 mL of co-solvent(MeOH:CH₂Cl₂ 1:1) was added sodium methoxide (0.024 mmol) and stirredfor 6 h under argon at ambient temperature. The reaction was neutralizedby Amberlite IR-120 and filtered. After removal of the solvent, theresidue was used for next step without prior purification.

Compound 23:

The hydrolyzed compound 22 was dissolved in 50 mL of aqueous acetic acidsolution (AcOH:H₂O 4:1) and stirred for 16 h at 60° C. After removal ofthe solvent, the mixture was purified by flash column chromatography onsilica gel (hexanes:AcOEt:MeOH 1:1:0.1).

Compound 24

The diacetonide derivative 23 was dissolved in 50 mL of co-solvent(MeOH:CHCl₃ 4:1) containing palladium hydroxide on carbon (20% Pd)(cat.) and stirred for 16 h in an H₂ atmosphere. The solution wasfiltered through Celite 545 to remove the catalyst and evaporated todryness, the mixture was purified by flash column chromatography onsilica gel (MeOH:CHCl₃ 1:9) and eluted with LH20 (MeOH:CHCl₃ 1:1) togive 24 (72 mg, 35% for three steps) as white solid. ¹H NMR (MeOD-CDCl₃1:1, 600 MHz) δ: 4.83 (s, 1H), 4.15 (d, J=4.2 Hz, 1H), 3.84 (dd, J=10.8,4.2 Hz, 1H), 3.76 (d, J=12.0 Hz, 1H), 3.64-3.70 (m, 2H), 3.60 (t, J=9.6Hz, 1H), 3.51-3.57 (m, 3H), 3.41 (d, J=9.6 Hz, 1H), 3.31 (m, 1H), 2.17(t, J=7.2 Hz, 2H), 1.50-1.65 (m, 4H), 1.19-1.39 (m, 68H), 0.85 (t, J=6.6Hz, 6H). ¹³C NMR (MeOD-CDCl₃ 1:1, 150 MHz) δ: 175.29, 100.06, 75.05,74.58, 73.03, 72.75, 72.62, 71.01, 67.78, 62.22, 51.11, 37.07, 32.93,32.62, 30.49, 30.45, 30.40, 30.35, 30.25, 30.13, 30.05, 30.04, 26.61,26.58, 23.34, 14.47. HRMS (MALDI-TOF) for C₅₀H₉₉NO₉Na⁺ [M+Na]⁺ calcd880.7223, found 880.7212.

Compound 25

Compound 25 was synthesized using the similar procedure as compound 24.¹H NMR (MeOD-CDCl₃ 1:1,600 MHz) δ: 7.09 (dd, J=8.4, 5.4 Hz, 2H), 6.90(t, J=9.0 Hz, 2H), 4.82 (d, J=3.6 Hz, 1H), 4.14-4.18 (m, 1H), 3.84 (dd,J=10.2, 4.8 Hz, 1H), 3.76 (dd, J=12.0, 2.4 Hz, 1H), 3.64-3.68 (m, 2H),3.60 (t, J=9.0 Hz, 1H), 3.51-3.56 (m, 3H), 3.41 (dd, J=9.6, 3.6 Hz, 1H),3.31 (m, 1H), 2.54 (t, J=7.8 Hz, 2H), 2.17 (t, J=7.8 Hz, 2H), 1.51-1.65(m, 6H), 1.20-1.39 (m, 36H), 0.85 (t, J=6.6 Hz, 3H). ¹³³³³C NMR(MeOD-CDCl₃ 1:1, 150 MHz) δ: 175.30, 162.67, 161.07, 139.18, 139.16,130.34, 130.29, 115.46, 115.32, 100.00, 75.03, 74.52, 72.97, 72.68,72.55, 70.93, 67.71, 62.15, 51.11, 51.03, 37.06, 37.00, 35.72, 32.90,32.58, 32.32, 30.45, 30.41, 30.35, 30.30, 30.22, 30.15, 30.11, 30.05,30.00, 29.82, 26.57, 26.53, 23.29, 14.44. HRMS (ESI-TOF) forC₄₁H₇₂FNO₉Na⁺ [M+Na]⁺ calcd 764.5083, found 764.5066.

Compound 26

Compound 26 was synthesized using the similar procedure as compound 24.¹H NMR (MeOD-CDCl₃ 1:1, 600 MHz) δ: 7.10 (d, J=8.4 Hz, 2H), 6.99 (t,J=7.8 Hz, 2H), 6.92 (m, 2H), 6.84 (d, J=7.8 Hz, 2H), 4.83 (d, J=3.0 Hz,1H), 3.85 (dd, J=10.2, 4.2 Hz, 1H), 3.77 (d, J=11.4 Hz, 1H), 3.67 (m,2H), 3.61 (t, J=9.6 Hz, 1H), 3.55 (m, 3H), 3.42 (dd, J=9.0, 3.0 Hz, 1H),3.33 (m, 1H), 2.55 (t, J=7.8 Hz, 2H), 2.18 (t, J=7.8 Hz, 2H), 1.50-1.64(m, 6H), 1.20-1.40 (m, 36H), 0.85 (t, J=6.6 Hz, 3H). ¹³C NMR (MeOD-CDCl₃1:1, 150 MHz) δ: 175.34, 160.27, 158.67, 156.22, 154.38, 138.73, 130.34,120.76, 120.71, 119.10, 116.87, 116.72, 100.15, 75.10, 74.66, 73.15,72.85, 72.65, 71.09, 67.80, 62.26, 51.21, 37.09, 35.90, 32.95, 32.68,32.44, 30.55, 30.51, 30.47, 30.40, 30.35, 30.28, 30.26, 30.17, 30.11,30.00, 26.67, 26.63, 23.38, 14.47. HRMS (ESI-TOF) for C₄₇H₇₆FNO₁₀Na⁺[M+H]⁺ calcd 834.5526, found 834.5538.

Synthesis of Fluorinated Donor 38

Compound 32:

To a solution of 1,2,3,4,6-Penta-O-acetyl-β-D-glucopyranose 1 in dryCH₂Cl₂ was added 4-methoxyphenol and BF₃OEt₂ at 0° C., the reaction wasstirred for 16 h at ambient temperature under argon. The resultingsolution was directly extracted with saturated NaHCO₃ solution andbrine, dried over MgSO₄ and evaporated. The product was recrystallizedfrom a solution of AcOEt-hexanes to give 32 as white solid.

Compound 33:

To a solution of 32 in dry MeOH was added catalytic amount of sodiummethoxide (NaOMe) and stirred for 3 h at ambient temperature. Thereaction was neutralized by adding Amberlite IR-120 and filtered, theresulting solution was concentrated to dryness to give 33 as whitesolid, which was directly used for next reaction without furtherpurification.

Compound 34:

To a solution of 33 in dry co-solvent (DMF and CH₃CN) was addedbenzaldehyde dimethylacetal and catalytic amount of sodium methoxide(NaOMe). The reaction was stirred for 16 h at ambient temperature. Thesolution was neutralized by adding Et₃N and concentrated. The mixturewas dissolved in ethyl acetate, washed with saturated NaHCO₃ solutionand brine, dried over MgSO₄ and evaporated. The product wasrecrystallized from a solution of AcOEt-hexanes to give 34 as whitesolid.

Compound 35:

To a solution of 34 in dry N,N-dimethylformamide (DMF) was added sodiumhydride (60% in mineral oil) at 0° C. The reaction was stirred for 1 h,followed by the addition of benzyl bromide and the reaction was stirredfor 16 h under argon at ambient temperature. The solution was quenchedby MeOH and evaporated to dryness. The residue was diluted with AcOEt,the solution was washed with H₂O and brine, dried over MgSO₄, andevaporated to dryness. The product was recrystallized from a solution ofAcOEt-hexanes to give 35 as white solid.

Compound 36:

To a solution of 35 in CH₂Cl₂ was added triethylsilane andtrifluoroacetic acid (TFA) at 0° C. The reaction was stirred for 3 h atambient temperature. The solution was directly washed with H₂O,saturated NaHCO₃ solution and brine, dried over MgSO₄, and evaporated todryness. The product was recrystallized from a solution of AcOEt-hexanesto give 36 as white solid.

Compound 37:

To a solution of 36 in CH₂Cl₂ was added diethylaminosulfur trifluoride(DAST). The reaction was stirred for 16 h at 45° C. and the solution wasdirectly washed with H₂O, saturated NaHCO₃ solution and brine, driedover MgSO₄, and evaporated to dryness. The residue was purified by flashcolumn chromatography on silica gel to give compound 37 as colorlessoil.

Compound 38:

To a solution of 37 in dry co-solvent (toluene, H₂O and CH₃CN) was addedceric ammonium nitrate (CAN). The reaction was stirred for 10 min atambient temperature. The solution was extracted with ethyl acetate,washed with H₂O, saturated NaHCO₃ solution and H₂O, dried over MgSO₄,and evaporated to dryness. The residue was purified by flash columnchromatography on silica gel to give compound 38 as colorless oil.

Synthesis of Fluorinated Analogue 43

Compound 39:

To a solution of donor 38, dimethylsulfide, 4 Å molecular sieve and2-chloropyridine in anhydrous CH₂Cl₂ was added trifluoromethanesulfonicanhydride at −45° C. under argon. The reaction was stirred for 20 min at−45° C., 20 min at 0° C. and another 20 min at ambient temperature,followed by the addition of acceptor 18 in CH₂Cl₂. The reaction wasstirred for 16 h at ambient temperature under argon. The solution wasfiltered through Celite 545 to remove molecular sieve. After removal ofthe solvent, the residue was diluted with AcOEt, the solution was washedwith H₂O and brine, dried over MgSO₄ and evaporated to dryness. Themixture was purified by flash column chromatography on silica gel togive 39.

Compound 40:

To a solution of 39 in pyridine/H₂O (10:1) was added triphenylphosphine.The reaction was stirred for 16 h at 45° C. under argon. After removalof the solvent, the residue was diluted with AcOEt, extracted with H₂O,brine and dried over MgSO₄ then evaporated to dryness. The mixture wasused for next step without prior purification.

Compound 41:

To a solution of compound 40 in anhydrous CH₂Cl₂ was added4-(4-fluorophenoxy) phenylundecanoic acid, Et₃N, EDC and HBTu. Thereaction was stirred for 16 h at ambient temperature under argon. Afterremoval of the solvent, the residue was diluted with AcOEt, extractedwith H₂O, brine and dried over MgSO₄ then evaporated to dryness. Themixture was purified by flash column chromatography on silica gel togive 41.

Compound 42:

To a solution of compound 41 in aqueous acetic acid solution (AcOH:H₂O4:1) and stirred for 16 h at 60° C. After removal of the solvent, themixture was dissolved in ethyl acetate, washed with saturated NaHCO₃solution and brine, dried over MgSO₄ and evaporated. The residue waspurified by flash column chromatography on silica gel to give 42.

Compound 43

The deacetonide derivative 42 was dissolved in co-solvent (MeOH:CHCl₃4:1) containing palladium hydroxide on carbon (20% Pd) (cat.) andstirred for 16 h in an H₂ atmosphere. The solution was filtered throughCelite 545 to remove the catalyst and evaporated to dryness, the mixturewas purified by flash column chromatography on silica gel and elutedwith LH20 to give 43. ¹H NMR (MeOD-CDCl₃ 1:1, 600 MHz) δ: 7.09 (1H, d,J=8.4 Hz), 6.96-6.99 (2H, m), 6.91-6.92 (2H, m), 6.82-6.84 (2H, m), 4.84(1H, t, J=3.6 Hz), 4.25 (0.5H, t, J=9.0 Hz), 4.15-4.18 (2H, m),3.82-3.85 (2H, m), 3.75-3.77 (1H, m), 3.69-3.72 (1H, m), 3.64-3.68 (2H,m), 3.50-3.54 (2H, m), 3.44 (1H, dd, J=9.6, 3.6 Hz), 2.54 (2H, t, J=7.2Hz), 2.17 (2H, t, J=7.8 Hz), 1.50-1.64 (7H, m), 1.22-1.27 (41H, m), 0.84(3H, t, J=7.2 Hz). ¹³C NMR (MeOD-CDCl₃ 1:1, 150 MHz) δ: 175.28, 160.20,158.61, 156.16, 154.30, 138.67, 130.28, 120.70, 120.64, 119.04, 116.81,116.66, 99.94, 90.61, 89.41, 75.04, 72.72, 72.59, 72.56, 72.42, 72.37,70.82, 70.66, 67.82, 61.21, 51.10, 37.05, 35.83, 32.91, 32.61, 32.36,30.46, 30.43, 30.39, 30.33, 30.27, 30.19, 30.18, 30.09, 30.03, 29.92,26.63, 26.55, 23.32, 14.41. HRMS (ESI-TOF) for C₄₇H₇₅F₂NO₉H⁺ [M+H]⁺calcd 836.5498, found 836.5483.

Synthesis of Fluorinated Donor 48

Compound 44:

To a solution of 32 in dry pyridine was added triphenylmethyl chloride.The reaction was stirred for 16 h at 60° C. under argon. After removalof the solvent, the mixture was purified by flash column chromatographyon silica gel to give 44.

Compound 45:

To a solution of 44 in N,N-dimethylformamide (DMF) was added sodiumhydride (60% in mineral oil) at 0° C. The reaction was stirred for 1 h,followed by the addition of benzyl bromide and stirred for 16 h underargon at ambient temperature. The reaction was quenched by MeOH andevaporated to dryness. The residue was diluted with AcOEt, the solutionwas washed with H₂O and brine, dried over MgSO₄, and evaporated todryness. The mixture was purified by flash column chromatography onsilica gel to give 45.

Compound 46:

To a solution of 45 in aqueous acetic acid solution (AcOH:H₂O 4:1) wasstirred for 3 h at 75° C. After removal of the solvent, the mixture wasdiluted with AcOEt, the solution was washed with H₂O and brine, driedover MgSO₄, and evaporated to dryness. The residue was purified by flashcolumn chromatography on silica gel to give 46.

Compound 47:

To a solution of 46 in CH₂Cl₂ was added diethylaminosulfur trifluoride(DAST). The reaction was stirred for 16 h at 45° C. and the solution wasdirectly washed with H₂O, saturated NaHCO₃ solution and brine, driedover MgSO₄, and evaporated to dryness. The residue was purified by flashcolumn chromatography on silica gel to give compound 47.

Compound 48:

To a solution of 47 in dry co-solvent (toluene, H₂O and CH₃CN) was addedceric ammonium nitrate (CAN). The reaction was stirred for 10 min atambient temperature. The solution was extracted with ethyl acetate,washed with H₂O, saturated NaHCO₃ solution and H₂O, dried over MgSO₄,and evaporated to dryness. The residue was purified by flash columnchromatography on silica gel to give compound 48 as colorless oil.

Synthesis of Fluorinated Analogue 53

Compound 49:

To a solution of donor 48, dimethylsulfide, 4 Å molecular sieve and2-chloropyridine in anhydrous CH₂Cl₂ was added trifluoromethanesulfonicanhydride at −45° C. under argon. The reaction was stirred for 20 min at−45° C., 20 min at 0° C. and another 20 min at ambient temperature,followed by the addition of acceptor 18 in CH₂Cl₂. The reaction wasstirred for 16 h at ambient temperature under argon. The solution wasfiltered through Celite 545 to remove molecular sieve. After removal ofthe solvent, the residue was diluted with AcOEt, the solution was washedwith H₂O and brine, dried over MgSO₄ and evaporated to dryness. Themixture was purified by flash column chromatography on silica gel(hexanes:AcOEt 10:1) to give 49.

Compound 50:

To a solution of 49 in pyridine/H₂O (10:1) was added triphenylphosphine.The reaction was stirred for 16 h at 45° C. under argon. After removalof the solvent, the residue was diluted with AcOEt, extracted with H₂O,brine and dried over MgSO₄ then evaporated to dryness. The mixture wasused for next step without prior purification.

Compound 51:

To a solution of compound 50 in dry CH₂Cl₂ was added 4-(4-fluorophenoxy)phenylundecanoic acid, Et₃N, EDC and HBTu. The reaction was stirred for16 h at ambient temperature under argon. After removal of the solvent,the mixture was diluted with AcOEt, extracted with H₂O, brine and driedover MgSO₄ then evaporated to dryness. The residue was purified by flashcolumn chromatography on silica gel to give 51.

Compound 52:

To a solution of compound 51 was dissolved in aqueous acetic acidsolution (AcOH:H₂O 4:1) and stirred for 16 h at 60° C. After removal ofthe solvent, the mixture was diluted with AcOEt, extracted with H₂O,brine and dried over MgSO₄ then evaporated to dryness. The residue waspurified by flash column chromatography on silica gel.

Compound 53

The deacetonide derivative 52 was dissolved co-solvent (MeOH:CHCl₃ 4:1)containing palladium hydroxide on carbon (20% Pd) (cat.) and stirred for16 h in an H₂ atmosphere. The solution was filtered through Celite 545to remove the catalyst and evaporated to dryness, the mixture waspurified by flash column chromatography on silica gel and eluted withLH20 to give 53. ¹H NMR (MeOD-CDCl₃ 1:1, 600 MHz) δ: 7.51 (0.6H, d,J=8.4 Hz), 7.09-7.11 (2H, m), 6.97-7.00 (2H, m), 6.91-6.94 (2H, m),6.83-6.85 (2H, m), 4.85 (1H, d, J=3.6 Hz), 4.49-4.59 (2H, m), 4.15-4.18(1H, m), 3.85 (1H, dd, J=10.8, 4.8 Hz), 3.61-3.71 (3H, m), 3.52-3.58(2H, m), 3.43 (1H, d, J=9.6, 3.6 Hz), 3.36 (1H, t, J=9.0 Hz), 2.55 (2H,t, J=7.8 Hz), 2.18 (2H, t, J=7.8 Hz), 1.51-1.64 (6H, m), 1.23-1.39 (39H,m), 0.85 (3H, t, J=7.2 Hz). ¹³C NMR (MeOD-CDCl₃ 1:1, 150 MHz) δ: 175.06,174.98, 159.99, 158.40, 155.94, 154.09, 154.08, 138.48, 130.09, 120.52,120.46, 118.85, 116.63, 116.47, 100.02, 83.18, 82.04, 74.72, 74.31,72.44, 72.38, 71.78, 71.67, 69.58, 69.54, 50.88, 50.79, 36.88, 36.83,35.64, 32.64, 32.42, 32.17, 30.29, 30.24, 30.20, 30.14, 30.08, 30.01,29.99, 29.91, 29.85, 29.74, 26.42, 26.38, 23.13, 14.26. HRMS (ESI-TOF)for C₄₇H₇₅F₂NO₉H⁺ [M+H]⁺ calcd 836.5483, found 836.5498.

Biological Studies Injection of Glycolipid Analogs in Mice

All the glycolipids were dissolved in 100% DMSO at a concentration of1-2 mg/ml. For in vivo experiments, all compounds were diluted to 10μg/ml in saline just before injection of 100 μl diluted glycolipid or100 μl 1% DMSO into mice. Pathogen-free C57BL/6 female mice aged 6-12weeks were obtained from the National Laboratory Animal Center (Taipei,Taiwan). Jα18 knockout (KO) B6 mice were the gifts from Dr. MasaruTaniguchi (RIKEN Research Center for Allergy and Immunology, Yokohama,Japan). All the mice were maintained in pathogen free vivarium ofInstitute of Cellular and Organismic Biology, Academia Sinica (Taipei,Taiwan).

Determination of Murine Cytokine/Chemokine Secretions

B6 WT or Jα18 KO mice were intravenously injected with vehicle orglycolipids at 0.1 or 1 μg/mouse. Serum was collected at 2 and 18 hafter injection for measurement of cytokines/chemokines by Beadlyte®Mouse Cytokine kit (Millipore, NY) and read by a Luminex® 200™ system(Luminex, Austin, Tex.).

FACS Analyses of Mouse Immune Cells after the Specific GlycolipidStimulation

B6 WT or Jα18 KO mice treated with specific glycolipid (1 μg/mouse) orvehicle (1% DMSO in PBS) were sacrificed at 72 hr post-injection andtheir spleens were harvested. After pressing spleens through 70 umstrainer and lysis of erythrocytes, the nucleated cells were resuspendedin PBS buffer containing azide (0.05%) and stained with antibodiesrecognizing the indicated cell surface antigens for 30 min at 4° C.After washing, the splenocytes were subjected to FACS analysis. Theantibodies against CD3, CD4, CD8α, CD11c, CD80, and CD86 were obtainedfrom BD Bioscience-Pharmingen.

Binding Strengths of the Binary Complex Between mCD1d and Glycolipid

Different concentrations of mCD1d^(di)-glycolipid complexes coated onthe ELISA plate were incubated with the saturated amounts of L363antibody (BioLegend) conjugated with biotin, followed bystreptavidin-HRP detection and ELISA measurement. The KD between L363antibody and the indicated mCD1d^(di)-glycolipid complex was calculatedfrom the linear regression of the Scatchard transformation of the L363antibody binding curve using GraphPad Prism software. L363 was found torecognize the mCD1d^(di)-7DW8-5-Glc complex and mCD1d^(di-)7DW8-5complex with similar binding strength. Next, the KD of the binarycomplex was determined as follows. Different concentrations ofglycolipids were incubated with fixed amounts of mCD1d dimer at 37° C.overnight, and then mCD1d^(di)-glycolipid complexes were coated on the96 well ELISA plate at 4° C. overnight. After washing and blocking withBSA at room temperature (RT) for 1 hr, L363 antibody conjugated withbiotin was added for 30 min at RT, followed by incubation withstreptavidin-HRP for 30 min at RT and detection with an ELSIA reader. KDvalues of the binary complex were calculated from the linear regressionof the Scatchard transformation of the L363 antibody binding curve.

Expansion of Human iNKT Cells

Human naïve Vα24⁺ iNKT cells were cultured with autologous immatureCD14⁺ DCs pulsed with the indicated glycolipid at 100 ng/ml or DMSO onday 2 for 18 h. On day 3, the suspension cells were transferred to a newdish, cultured in the presence of 50 U/ml IL-2 (R & D Systems), andreplenished with fresh medium every 3 days. The percentage ofVα24⁺/Vβ11⁺ cells was determined by flow cytometry on day 9. The totalcell number after expansion was calculated with the Guava ViaCountreagent (Millipore, USA) and detected by the Guava system with CytoSoft™software containing the ViaCount module (Millipore, USA).

Binding Avidity of Various CD1d-Loaded Glycolipids to Vα14+ iNKT Cells

Briefly, murine CD1d:Ig dimer (BD Biosciences PharMingen, San Diego,Calif.) was loaded with glycolipids at a molar ratio of 1:10 or vehiclefor overnight at 37° C. Murine 1.2 Vα14⁺ iNKT cells were incubated withvarious doses of dimer-glycolipid complex in buffer containing azide(0.05%) for 30 min at 4° C. These cells were then stained withanti-mouse IgG1-PE mAb (A85-1) for another 30 min at 4° C., followed bywashing, fixation with 4% paraformaldehyde (PFA), and the bound mCD1ddimer complexes were detected by flow cytometry. The binding curve andlinear fit of the Scatchard transformation were plotted by GraphpadPrism software.

Binding Avidity of CD1d-Loaded Glycolipids with Vα24+ iNKT Cells

Binding avidity of human CD1d-glycolipid complexes to Vα24⁺ iNKT cellsexpanded by 7DW8-5 at 100 ng/ml was determined as describedpreviously.¹⁹

Isolation and Generation of Human Vα24+ iNKT Cell Lines and ImmatureMonocyte-Derived Dendritic Cells

Vα24⁺ iNKT cells and CD14⁺ cells were isolated from peripheral bloodcells as described previously.¹⁹ Immature DCs were generated from theCD14⁺ cells after 2-day incubation in the presence of 300 U/ml GM-CSF (R& D Systems) and 100 U/ml IL-4 (R& D Systems).

Vα24⁺ iNKT cell lines expanded with 7DW8-5 or C1 were generated asfollows. After irradiation with 2,000 rad, the immature DCs werecocultured with syngenic Vα24⁺ iNKT cells in the presence of 7DW8-5 orC1 at 100 ng/ml for 1 day. The cells were expanded in the presence of 50U/ml IL-2 for 10-14 days after lipid removal. The same procedures wererepeated once for further stimulation and expansion of iNKT cells. The7DW8-5 or C1-expanded iNKT cell line was shown to express Vα24 T cellantigen receptor (>95% purity).

mCD1d Vs. hCD1d Swapping Assay

Murine DN3A4-1.2 Vα14⁺ iNKT hybridoma cells or C1-expanded Vα24⁺ iNKTcells were pulsed with the indicated glycolipid antigen presented eitherby mCD1d (A20-CD1d cells) or hCD1d (HeLa-CD1d cells) at 1, 0.1, and 0.01μg/ml. After 18 hr, the supernatants were harvested for the measurementof cytokine secretion(s). IL-2 released from Vα14⁺ iNKT cells wasdetermined by ELISA assay. IFN-γ, IL-4 and IL-2 secreted from Vα24⁺ iNKTcells were detected using Beadlyte® Human Cytokine kit (Millipore, NY,USA) and Luminex® 200™ reading system.

Computer Modeling and Simulation

The crystal structures of both human and mouse CD1d (hCD1d and mCD1d)presenting α-GalCer to their respective iNKT TCRs were retrieved fromthe RCSB Protein Data Bank (www.rcsb.org; PDB coded 3HUJ, 3QUX, 3QUY,3QUZ, and 3HE6). These crystal structures were superimposed byreferencing to the backbone atoms of 3QUX. The two α-GalCer ternarystructures obtained from 3HE6 and 3HUJ were used to create the otherternary complexes composed of different GSLs. The ternary structurecontaining C34 was derived from that containing C1 by modifying the acylchain on Meastro (Schrödinger LLC, USA). The ternary structures bearingC1-Glc and C34-Glc were created by a inverting the O4 chirality of C1and C34, respectively. The modeling for the remaining new ternarycomplexes were built using these GSLs and CD1d-iNKT TCR structures from3QUX and 3HUJ for mice and humans, respectively. All structures wereprocessed using Protein Preparation wizard (Schrödinger LLC, USA) andthe lipid tails were further refined to minimize the steric collisionusing MacroModel's conformation search and energy minimization with thedefault methods and OPLS2005 force field in a solvent of water. Thebinding modes of GSLs to iNKT TCRs were recomputed by Autodock4.2 usinga semi-empirical free energy force field to evaluate conformationsduring simulations. The estimated free energy of binding in solvent wascomputed by the equation built in the Autodock4.2:

ΔG=ΔV ^(L)(bound−undound)+ΔV ^(P)(bound−undound)+ΔV^(PL)(bound−undound+ΔS _(conf)).

where L refers to the “ligand”; P refers to the “protein”; V refers tothe pair-wise energetic terms including evaluations fordispersion/repulsion, hydrogen bonding, and electrostatics; ΔS_(conf) isan estimate of the conformational entropy lost upon binding. Allrequired input files for running Autodock4 were prepared on MGLTools. Agrid box of 60×60×60 Å³ and various atom-typed energy maps weregenerated. In each molecular docking run, all hydroxyl groups were setto free rotation and the two lipid tails were assigned to their priorrefined poses. The Lamarckian genetic algorithm (maximum number of5.0×10⁷ energy evaluations and 27,000 generations and a mutation rate of0.02 with a crossover rate of 0.8) was employed for search. The resultswere visualized in MGLTools and the contribution of hydrogen bonding toindividual residue was obtained by the built in function. Graphicrepresentation was finished on Maestro.

Results

Glycosphingolipids (GSLs) with αGlc are Stronger Immune Modulators thanThose with αGal in Humans but Weaker in Mice

Previously, 0.1 μg/mouse of 7DW8-5 was sufficient for immunestimulation,¹⁹ but higher dosage (1 μg/mouse) was required for theglucose analog 7DW8-5-Glc to induce immune responses (FIG. 6). Thus, thebiological activities of newly synthesized glycolipids were tested in B6mice 2 and 18 hr after i.v. injection of glycolipids at 1 μg/mouse. Asshown in FIG. 2 and FIG. 7, the mannose analog 7DW8-5-Man failed toinduce any cytokines/chemokines. As compared to GSLs with αGlc, GSLswith αGal induced higher levels of cytokines and chemokines, includingIFN-γ, IL-2, IL-4, IL-6, GM-CSF, TNFα and IP-10. A similar trend wasnoted for αGalCer and C34 analogs with different glycosyl groups (FIG.2A, 2B and FIG. 7).

Although C1-Glc and C34-Glc triggered both cytokines and chemokines,7DW8-5-Glc induced very low levels of Th1 and Th2 cytokines butrelatively high levels of KC, MCP-1, IP-10 and MIG chemokines. To probethe possibility that other immune cells than iNKT cells might contributeto the chemokine induction by 7DW8-5-Glc, Jα18 KO mice which harbored noiNKT cells were injected with 7DW8-5-Glc or 7DW8-5 at 1 μg/mouse.²⁵Mouse sera collected 2 and 18 hr after injection showed no induction ofcytokines by either 7DW8-5 or 7DW8-5-Glc (FIG. 2A, 2B and FIG. 7).Surprisingly, 7DW8-5-Glc but not 7DW8-5 triggered the secretion ofseveral chemokines including KC, MCP-1, IP-10 and MIG in Jα18 KO mice.These findings suggested that immune cells other than iNKT cells in Jα18KO mice must have contributed to the production of chemokines in WT miceand Jα18 KO mice treated with 7DW8-5-Glc.

Next, we analyzed the expansion/activation of immune cells in WT mice 3days after glycolipid stimulation. The numbers of total T cells, CD4⁺ Tand CD8⁺ T cells were higher in mice treated by GSLs with αGal head thanthose treated by GSLs with αGlc (FIGS. 2C, 8E and 8F). This was in linewith the observation that more cytokines/chemokines were induced by GSLswith αGal than GSLs with αGlc in mice (FIGS. 2A, 2B and 7). Furthercomparison of immunostimulatory activities among GSLs with αGlc revealedthat both C1-Glc and C34-Glc were better than 7DW8-5-Glc in theinduction of cytokines/chemokines (FIGS. 2A, 2B and 7) and theexpansion/activation of DCs (FIG. 8B-8D). C34-Glc activated ˜2 fold moreCD80⁺ or CD86⁺ DCs than 7DW8-5-Glc (FIGS. 8C and 8D) although theyinduced similar numbers of total splenocytes and DCs (FIGS. 8A and 8B).As compared to 7DW8-5-Glc, C1-Glc not only expanded ˜1.3 fold moresplenocytes and DCs (FIGS. 8A and 8B) but also activated ˜3.5 fold moreCD80+ DCs as well as ˜3 fold more CD86⁺ DCs (FIGS. 8C and 8D). Theincreased expansion/activation of DCs may contribute to the strongerimmunogenicity triggered by C1-Glc and C34-Glc as compared to 7DW8-5-Glcin vivo. In contrast, 7DW8-5-Man did not expand any types of immunecells (FIGS. 2C and 8), consistent with the lack of induction ofcytokines/chemokines (FIGS. 2A, 2B and 7).

Unexpectedly, we also observed that 7DW8-5-Glc induced chemokines inJα18 KO mice (FIG. 7). FACS analyses of Jα18 KO mouse splenocytes 3 daysafter i.v. injection of 7DW8-5-Glc or 7DW8-5 revealed that CD11c^(hi)monocyte-derived DCs were significantly expanded and activated by7DW8-5-Glc but not 7DW8-5 (FIGS. 9B-9D). No significant differences inthe total splenocytes, CD4⁺ T and CD8⁺ T cells were noted afterstimulation with either 7DW8-5 or 7DW8-5-Glc (FIGS. 9A, 9E and 9F).These findings indicated that monocytes might be responsible for theinduction of chemokines such as KC, MCP-1, IP-10 and MIG in Jα18 KO micetreated with 7DW8-5-Glc.

As described above, glycolipid analogs with αGal were stronger immunemodulators than those with αGlc in mice, especially for the comparisonbetween 7DW8-5 and 7DW8-5-Glc. To investigate if similar trends alsoapplied to human iNKT cells, Vα24⁺ iNKT cells isolated from theperipheral blood were incubated with immature DCs pulsed with theindicated glycolipid at 100 ng/ml on day 2. After antigen removal on day3, human iNKT cells were cultured in the presence of IL-2. The number ofexpanded iNKT cells was counted using the Guava ViaCount reagent on day9. Surprisingly, 7DW8-5-Glc was significantly (p=0.0009) better than7DW8-5 in expanding human iNKT cells in vitro (FIG. 2D). Taken together,these findings suggested that the bioactivities of GSLs with αGal weremore potent in mice but less in humans as compared to GSLs with αGlc.

Binary Interaction Between mCD1d and Glycolipids

To understand the basis for the differences in the immune modulatingactivities of 7DW8-5 and 7DW8-5-Glc, we measured the binding strength ofthe binary interaction between mCD1d and specific glycolipid using L363mAb which could bind to mCD1d complexed with glycolipids.²⁶ Variousconcentrations of mCD1d^(di)-glycolipid complexes at fixed ratio wereincubated with saturated amounts of L363-biotin antibody, followed bystreptavidin-HRP detection and ELISA measurement (FIG. 10A). Thedissociation constant (KD) between L363 and the indicatedmCD1d^(di)-glycolipid complexes was calculated from the linearregression of the Scatchard transformation of the plot (FIG. 10A) usingGraphPad Prism software. L363 was found to recognizemCD1d^(di)-7DW8-5-Glc complex with similar binding strength as withmCD1d^(di-)7DW8-5 complex (FIG. 10B). Next, we determined the KD of thebinary complex by incubating different concentrations of glycolipidswith fixed amounts of mCD1d dimer and L363-biotin antibody, followed bystreptavidin-HRP detection and ELISA measurement (FIG. 10C). The KD wascalculated from the Scatchard transformation of the binding curve drawnfrom the L363 detection readout (FIG. 10D). Not surprisingly, 7DW8-5-Glchaving identical lipid tails as 7DW8-5 bound to mCD1d dimer with similarstrength, but their binding avidities were ˜20 fold greater thanαGalCer. This indicated that the strength of the binary interactioncould not account for the differential immune activating capacitiesbetween 7DW8-5 and 7DW8-5-Glc.

Ternary Interaction Between CD1d-GSL Complexes and iNKT Cells

Next, we measured the ternary interaction between CD1d-glycolipidcomplex and the iNKT TCR in mice and humans. Different concentrations ofmCD1d^(di)-glycolipid and hCD1d^(di)-glycolipid complexes were incubatedwith fixed amounts of DN3A4-1.2 murine iNKT hybridoma cells and humanVα24+/Vβ11⁺ iNKT cells, respectively. The level of bound complexes atthe indicated concentration was detected by anti-mIgG1 secondaryantibody and analyzed by flow cytometry (FIGS. 3A and 3B). The KD of theternary complex was calculated from the Scatchard transformation of theplots in FIGS. 3A and 3B using GraphPad Prism software. As shown in FIG.3C, mCD1d-7DW8-5 complex displayed ˜10-fold stronger interaction withiNKT TCR than mCD1d-7DW8-5-Glc complex. This was consistent with theobservation that higher percentages of C1-pulsed splenocytes werestained by the mCD1d-7DW8-5 complex (36.2±5.0%) than mCD1d-7DW8-5-Glccomplex (17.1±0.8%) (FIG. 11). When complexed with mCD1d, both C1 (KD:1.240±0.003 nM) and C34 (KD: 0.735±0.010 nM) exhibited stronger ternaryinteractions toward iNKT TCR than C1-Glc (KD: 5.137±0.110 nM) andC34-Glc (KD: 7.960±1.269 nM), respectively (FIG. 3C).

In humans, GSLs with αGlc (KD of C1-Glc: 8.550±0.617; C34-Glc:0.378±0.019; 7DW8-5-Glc: 0.481±0.008 nM) exhibited stronger ternaryinteractions toward Vα24+/Vβ11⁺ iNKT TCR than GSLs with αGal (KD of C1:16.410±4.200; C34: 0.498±0.005; 7DW8-5: 0.777±0.022 nM) in complex withhCD1d (FIG. 3D). Thus, irrespective of the types of lipid tails, GSLswith αGal exhibited stronger ternary interaction with mouse iNKT TCR butweaker ternary interaction with human iNKT TCR than GSLs with αGlc(FIGS. 3C and 3D). This may account for the observation that GSLs withαGal triggered higher levels of cytokines/chemokines and greaterexpansions of immune cells in mice (FIGS. 2A˜2C, 7 and 8) while lessiNKT cell expansion in humans (FIG. 2D) Taken together, the ternaryinteraction among iNKT TCR, CD1d and GSL seems to be more relevant forthe bioactivities of glycolipids than the binary interaction betweenCD1d and GSL.

Effects of Swapping Human Vs. Mouse CD1d Molecules Against iNKT Cells

To explain the species-specific responses, we examined the effects ofswapping human vs. mouse CD1d molecules against human vs. murine iNKTcells on the stimulatory activities of GSLs with different glycosylgroups. Murine iNKT hybridoma cells (FIG. 4A) or C1-expanded human Vα24⁺iNKT cells (FIG. 4B) were pulsed with the indicated glycolipid presentedby either mCD1d (A20-CD1d) or hCD1d (HeLa-CD1d). The supernatants wereharvested 24 hr later to measure the cytokine secretions. Whetherpresented by mCD1d or hCD1d, GSLs with αGal induced more IL-2 secretionthan GSLs with αGlc from murine iNKT cells (FIG. 4A). This wasconsistent with the in vivo findings that GSLs with αGal were morepotent than GSLs with αGlc to induce serum cytokine secretion (FIGS. 2and 7). In contrast, when presented by either mCD1d or hCD1d, GSLs withαGlc triggered more IL-2 secretion than GSLs with αGal from human iNKTcells (FIG. 4B). Similar trends were also observed on IFN-γ and IL-4secretions from human iNKT cells (FIG. 12A and FIG. 12B). Thespecies-specific stimulatory activities of GSLs with different glycosylgroups were dictated by the murine vs. human iNKT TCR, rather than CD1d.In comparison, 7DW8-5-Man could not stimulate mouse and human iNKT cellsto secret any cytokines regardless of its presentation by mCD1d orhCD1d. Notably, all GSLs with αGlc triggered significantly moreTh1-skewed responses than C1 based on the ratio of IFN-γ over IL-4 (FIG.12C). Besides, irrespective of lipid tails, GSLs with αGlc seemed moreTh1-biased than GSLs with αGal in humans (FIG. 12C). These findingsindicated that modification at the 4′-OH of the glycosyl group couldselectively induce the responses of human iNKT cells toward Th1direction.

Structural Modeling of the Ternary Complex of CD1d-GSL-iNKT TCR

To further explain differential binding avidities of the ternary complexin mice and men, computer modeling was performed based on the x-raystructures of murine and human CD1d-αGalCer-iNKT TCR complexes,respectively (PDB access code 3HUJ, 3QUX, 3QUY, 3QUZ, and 3HE6).²⁷⁻²⁹

(1) Interactions of the Sugar Head Groups

As shown in FIGS. 3A and 3B, the GSL with Man could not bind to themouse and human iNKT cells when complexed with CD1d. A vertical 2′OH inmannose created a steric hindrance against iNKT TCR (Ser30 in men andAsn30 in mice) and lost two hydrogen bonds originally formed by the 2′OHof galactose toward the iNKT TCR (Gly96 and Asp 151 in men as well asGly96 and Asp153 in mice). As for GSLs with Gal, the binding of C1 toCD1d and iNKT TCR for mice and humans was shown in FIGS. 5A and 5B,respectively. Formations of hydrogen bond (H-bond) interactions wereobserved in most conserved residues, including human Asp80 (mouseAsp80), human Thr154 (mouse Thr156), human Asp151 (mouse Asp 153) ofCD1d and human Gly96 (mouse Gly96) of iNKT TCR. On the other hand, theH-bond interactions of the 3′OH/4′OH of C1 with human and mouse iNKT TCRwere quite different. The residue Asn30 of mouse iNKT TCR was crucialfor binding to the 3′- and 4′-OHs of C1. The free energy contribution ofAsn30 was estimated to be −2.27˜−3.38 Kcal/mol using MGLTools. Incomparison, Ser30 of human iNKT TCR was more distant from the 4′-OH ofC1, resulting in a weaker H-bond interaction with the 3′-OH only, whilea H-bond could be formed between 4′-OH of C1 and the backbone C═O groupof Phe29 (FIG. 5B). The free energy contribution of Ser30 with the 3′-OHand Phe29 with the 4′-OH of C1 was computed to be about −1.23˜−1.63Kcal/mol. Thus, a change from axial (Gal) to equatorial (Glc) directionof 4′ OH would lose the H-bond interaction with mouse Asn30 and humanPhe29 of iNKT TCRs. As compared to C1 and C34, respectively, C1-Glc andC34-Glc lacked the H-bond interaction with murine Asn30, leading to adecreased free energy (−0.7˜−0.9 Kcal/mol calculated by MGLTools). Thiswas in line with the drops in the murine ternary interaction in the KDmeasurement (FIG. 3C) when galactose was changed to the glucose head. Onthe contrary, human ternary interactions were greater for GSLs withglucose (FIG. 3D). Based on the computer modeling, we found that theequatorial 4′-OH of glucose could compensate for the loss of Phe29interaction (−0.4 Kcal/mol) by a stronger interaction (˜−1.84 Kcal/mol)with a crystal water, which was trapped by human iNKT TCR-Phe51 andhCD1d-Trp153 (FIG. 5C). Without the formation of hydrophobic space andthe trapped water molecule in mice, the ternary interaction would beweaker for GSLs with Glc than those with Gal.

(2) Interactions of the Lipid Tails

The two aromatic rings at the acyl tail of C34 could form aromaticinteractions with Phe70 and Trp63 of CD1d. Thus, the change of the acyltail from C1 to C34 could increase the interaction (−1.8˜−2.4 Kcal/molby modeling) with CD1d. In addition, the higher energy from aromaticinteractions could drive the acyl chain of C34 or C34-Glc to a lowerposition (near Cys12) of the A′ channel within CD1d, leading to a subtleperturbation to the orientation of the head group (FIG. 5D). Therefore,without a congenial force between mouse iNKT TCR and the equatorial4′-OH of the glucose head, the binding of mCD1d-C34-Glc to iNKT TCR wasa little weaker than mCD1d-C1-Glc (FIG. 3C). This may explain whyC34-Glc was less potent than C1-Glc while C34 was superior to C1 inmice.

(3) Computed Free Energy Using AUTODOCK4

The free energy of each GSL bound to human and mouse CD1d-iNKT TCRs wascomputed in triplicate. In each round, the GSLs were docked to the humanand mouse CD1d-C1-iNKT TCRs and the topmost ranked free energies wereselected. As shown in FIG. 5E, the computed free energy in generalcorrelated with the trends of the KD values measured for mice (FIG. 3C)and humans (FIG. 3D).

A variety of means can be used to formulate the compositions of theinvention. Techniques for formulation and administration may be found in“Remington: The Science and Practice of Pharmacy,” Twentieth Edition,Lippincott Williams & Wilkins, Philadelphia, Pa. (1995). For human oranimal administration, preparations should meet sterility, pyrogenicity,and general safety and purity standards comparable to those required bythe FDA. Administration of the pharmaceutical formulation can beperformed in a variety of ways, as described herein.

Cytokine Induction and Ternary Interactions Among GSLs, CD1d and iNKTTCR

As compared to αGalCer, αGlcCer has been reported to be less stimulatoryon the murine iNKT cell proliferation but appeared more potent tostimulate human iNKT cell proliferation.^(5,22) In our studies, thedifferential activities between αGalCer and αGlcCer in mice and humanswere also observed on the cytokine induction and ternary interactionamong GSLs, CD1d and iNKT TCR. Furthermore, phenyl GSLs bearing αGal orαGlc head showed similar species-specific activities. Thus, irrespectiveof lipid tails, GSLs with αGal were better than GSLs with αGlc in micebut worse in men. This indicated that the bioactivity of glycolipids inmice cannot be translated to that in humans despite the highlyhomologous sequences of CD1d and iNKT TCR between mice and humans.³⁰

The species-specific responses were most likely attributed to thedifferences in iNKT TCR between mice and men, as demonstrated by mCD1dvs. hCD1d swapping assay. GSLs with αGal, either presented by mCD1d orhCD1d, were more potent than GSLs with αGlc to stimulate murine iNKTcells but less stimulatory for human iNKT cells. According to thecrystal structures of CD1d-αGalCer-iNKT TCR, the residues in contactwith the 4′-OH of the galactose head in the CDR1α region of iNKT TCRwere not conserved between mice (Asn30) and humans (Phe29).^(27,28) Ourcomputer modeling revealed that the change of 4′-OH from the axial toequatorial direction could result in the loss of hydrogen bond betweenthe 4′OH and iNKT TCR-Asn30 in mice. On the contrary, the equatorial4′-OH of glucose could compensate for the loss of Phe29 interaction by astronger interaction with a crystal water, which was trapped by humaniNKT TCR-Phe51 and hCD1d-Trp153. Thus, the ternary interaction composedof GSLs with αGlc was stronger than GSLs with αGal in humans but weakerin mice. Taken together, the structural modeling of murine and humanCD1d-GSL-iNKT TCR complexes provided a good explanation forspecies-specific biological activities, and the computed free energychanges correlated well with the trends of measured binding avidities ofthe ternary complex.

In contrast to our paired analogues with the same tail but differentglycosyl groups, the species-specific immune responses were also seen inother cases with the same glycosyl group but different lipidtails.^(31,32) Similarly, it was the iNKT TCR, instead of CD1d, thatshaped the preferential activities of two C-glycoside analogs betweenmice and men, which correlated well with the binding strengths of theternary complex.³¹ For our glycolipids, ternary interaction was alsomore important than binary interaction in predicting the biologicalresponses in mice and men. Both 7DW8-5 and 7DW8-5-Glc bound with mCD1dmuch stronger than C1, probably due to the increased contacts of thephenyl ring and the fluoride atom with the CD1d A′ pocket.³³ Even thoughthe two glycolipids with the same lipid tails exhibited similar bingingstrengths toward CD1d, they showed different immune stimulatorypotencies. 7DW8-5-Glc was better than 7DW8-5 in men but worse in mice,which had a good relationship with the binding avidities of ternarycomplexes. In addition to 7DW8-5 and 7DW8-5-Glc, the bioactivities ofthe other two paired analogues (C1 vs. C1-Glc and C34 vs. C34-Glc) alsocorrelated well with the strengths of the ternary interaction in miceand men. That is, GSLs with αGal showed stronger ternary interaction andimmune stimulatory activities than GSLs with αGlc in mice, but the trendwas opposite in men. Hence, the measurement of the ternary interactionin vitro could be used to predict the immune-stimulatory potency of ournew glycolipids in vivo.

Further comparison among GSLs with αGlc revealed that both C1-Glc andC34-Glc were better than 7DW8-5-Glc in cytokine induction in mice.However, the binding avidities of the murine ternary complex werecomparable for C34-Glc and 7DW8-5-Glc. These findings suggested thatfactors other than KD may also modulate immune responses in vivo. Infact, both C1-Glc and C34-Glc activated more CD80⁺ or CD86⁺ DCs than7DW8-5-Glc, which may contribute to the stronger bioactivities of C1-Glcand C34-Glc in mice. Taken together, several factors, including thestrength of the ternary interaction and the expansion/activation ofimmune cells, could regulate immune stimulation in vivo.

In contrast to GSLs with αGal or αGlc, αManCer and 7DW8-5-Man failed toinduce iNKT cell proliferation,^(5,22) cytokines/chemokines, and/or theexpansion/activation of immune cells in both mice and humans. This maybe attributed to the fact that neither murine nor human iNKT TCR couldrecognize the αMan head, as demonstrated by the lack of staining withCD1d-7DW8-5-Man dimer in iNKT cells. As compared to 7DW8-5-Man,7DW8-5-Glc was able to induce Th1 and Th2 cytokines albeit at very lowlevels in mice. In comparison, large amounts of KC, MCP-1, IP-10 and MIGchemokines were triggered by 7DW8-5-Glc in WT and Jα18 KO mice,indicating that certain types of immune cells other than iNKT cells maycontribute to these chemokine secretions. Indeed, monocytes weresignificantly expanded/activated in Jα18 KO mice by 7DW8-5-Glc. It hadbeen reported that monocytes could produce these chemokines in responseto stimulations,³⁴⁻³⁷ suggesting that monocytes may be responsible forchemokine secretions in 7DW8-5-Glc-treated mice. Nevertheless, we couldnot exclude that other possible sources existing in Jα18 KO mice mayalso play a role. Vα10 NKT cells could produce IFN-γ and IL-4 inresponse to αGalCer and αGlcCer in vitro,³⁸ but IFN-γ was not secretedin the sera of Jα18 KO mice treated with αGalCer.³⁹ We could not detectany cytokine productions from Jα18 KO mice treated with 7DW8-5 or7DW8-5-Glc. These findings implied that Vα10 NKT cells contributedlittle to the chemokines triggered by 7DW8-5-Glc in mice.

However, most of the cytokines and chemokines, including IFN-γ, IL-2,IL-4, IL-6, GM-CSF and TNFα were not produced in Jα18 KO mice stimulatedwith either 7DW8-5 or 7DW8-5-Glc. Immune cells like CD4⁺ T and CD8⁺ Tcells were not expanded in Jα18 KO mice either. Thus, iNKT cellsremained to be the key player in the above-mentioned cytokine/chemokineinduction and immune cell expansion by 7DW8-5 or 7DW8-5-Glc.

In summary, GSLs with αGlc bore stronger ternary interaction andtriggered more Th1-biased immunity as compared to GSLs with αGal inhumans. However, GSLs with αGlc were less stimulatory than GSLs withαGal in mice. The species-specific responses were attributed to thedifferential binding avidities of ternary complexes between species,reflecting the differences between murine and human iNKT TCR assupported by mCD1d vs. hCD1d swapping assay. This was in line with theprediction by the computer modeling based on the crystal structures ofthe CD1d-αGalCer-iNKT TCR complex in mice and men.²⁷⁻²⁹ In addition tothe ternary interaction between CD1d-glycolipid complex and iNKT TCR,expanded/activated monocytes could also modulate immune responses invivo, especially for GSLs with αGlc.

From our studies, the change of the 4′OH direction on the glycosyl groupled to different bioactivities in mice and humans. This was consistentwith the report that the aromatic group introduced to 4′ OH of theαGalCer head could affect iNKT cell cytokine production in mice, buttheir effects in humans were not investigated.⁴⁰ Alterations at the 6position of the glycosyl group also showed variable effects on thebiological responses.^(29,41) These findings together with our workprovided a new direction for the future design and synthesis of newGSLs.

Throughout this application, various publications, patents and publishedpatent applications are cited. The inventions of these publications,patents and published patent applications referenced in this applicationare hereby incorporated by reference in their entireties into thepresent invention. Citation herein of a publication, patent, orpublished patent application is not an admission the publication,patent, or published patent application is prior art.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claim.

We claim:
 1. A human immune adjuvant compound having Formula (I):

or a pharmaceutically acceptable salt thereof; wherein: R¹ is —OH orhalogen; R² is —OH or halogen; R³ is hydrogen; R⁴ is selected from thegroup consisting of optionally substituted carbocyclyl, optionallysubstituted aryl, optionally substituted heterocyclyl, optionallysubstituted heteroaryl, optionally substituted alkoxy, an optionallysubstituted amino group, and optionally substituted acyl, R⁵ is selectedfrom the group consisting of hydrogen, halogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted aryl,optionally substituted heterocyclyl, optionally substituted heteroaryl,optionally substituted alkoxy, an optionally substituted amino group,and optionally substituted acyl; n is an integer of 1 to 15, inclusive;and m is an integer of 1 to 20, inclusive.
 2. The compound of claim 1,wherein R² is —OH, and R¹ is halogen.
 3. (canceled)
 4. The compound ofclaim 1, wherein R¹ is —OH, and R² is halogen.
 5. (canceled)
 6. Thecompound of claim 1, wherein R⁴ is of Formula (II):

wherein: i is 0, 1, 2, 3, 4, or 5; R⁶ is independently selected from thegroup consisting of hydrogen, halogen, —CN, —NO₂, —N₃, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted aryl, optionally substituted heterocyclyl, optionallysubstituted heteroaryl, optionally substituted alkoxy, an optionallysubstituted amino group, and optionally substituted acyl.
 7. Thecompound of claim 6, wherein R⁶ is halogen.
 8. The compound of claim 7,wherein the halogen is F.
 9. The compound of claim 1, wherein R⁴ is ofFormula (III):

wherein: j is 0, 1, 2, 3, or 4; k is 0, 1, 2, 3, 4, or 5; and eachinstance of R⁷ and R⁸ is independently selected from the groupconsisting of hydrogen, halogen, —CN, —NO₂, —N₃, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted aryl,optionally substituted heterocyclyl, optionally substituted heteroaryl,optionally substituted alkoxy, an optionally substituted amino group,and optionally substituted acyl.
 10. The compound of claim 9, whereineach instance of R⁷ and R⁸ is independently hydrogen or halogen.
 11. Thecompound of claim 9, wherein R⁷ is hydrogen; R⁸ is F; and k is 1, 2 or3.
 12. The compound of claim 9, wherein R⁷ is F; R⁸ is hydrogen; and jis 1, 2 or
 3. 13. The compound of claim 9, wherein R⁷ and R⁸ both are F;k is 1, 2 or 3; and j is 1, 2 or
 3. 14. The compound of claim 1, whereinthe compound is selected from one of the following:


15. A pharmaceutical composition comprising: (i) a compound according toany one of claims 1 to 14 in an amount sufficient to stimulate an immuneresponse when co-administered with an antigen to a human subject, and(ii) a pharmaceutically acceptable excipient.
 16. A method foraugmenting an immunogenicity of an antigen in a subject in need thereof,comprising co-administering said antigen with an adjuvant compositioncomprising a compound of claim
 1. 17. A method for stimulating an immuneresponse in a human subject in need thereof, the method comprising:administering to the subject a therapeutically effective amount of animmune adjuvant composition in a pharmaceutically acceptable carrier,wherein the composition comprises a compound according to any one ofclaims 1-14.
 18. The method of claim 16, wherein the adjuvantcomposition is a vaccine adjuvant.
 19. The method of claim 16, whereinthe adjuvant composition is administered in an amount capable ofelevating the level of invariant Natural Killer T (iNKT) cells inhumans.
 20. The method of claim 19, wherein administration of theadjuvant composition increases production of one or more cytokinesand/or one or more chemokines in humans.
 21. The method of claim 20,wherein the cytokine production is sufficient to transactivatedownstream immune cells.
 22. The method of claim 21, wherein thedownstream immune cells comprise one or more of dendritic cells (DC),natural killer cells (NK), B cells, CD4⁺ T and CD8⁺ T cells.
 23. Themethod of claim 20, wherein the cytokines comprise Th1 cytokines. 24.The method of claim 23, wherein the Th1 cytokine is selected from atleast one of the group consisting of interferon-gamma (IFN-γ), GM-CSF,TNFα, interleukin 2, interleukin 12 and interleukin
 10. 25. The methodof claim 20, wherein the chemokine is selected from at least one of thegroup consisting of RANTES, MIP-1α, KC, MCP-1, IP-10 and MIG.
 26. Themethod of claim 16, wherein administration of the composition results inan anti-cancer effect.
 27. The method of claim 26, wherein the cancer isselected from the group consisting of lung cancer, breast cancer,hepatoma, leukemia, solid tumor and carcinoma.
 28. The method of claim16, wherein R⁴ in the compound of Formula I is selected from substitutedor unsubstituted aryl, and substituted or unsubstituted heteroaryl, andwherein increase in Th1 cytokines in humans exceeds any increase in Th2cytokines.
 29. A method for elevating the level of invariant NaturalKiller T (iNKT) cells production in a human subject in need thereof, themethod comprising: administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition, wherein thecomposition comprises a compound according to any one of claims 1 to 14.30. The method of claim 29, wherein the elevation of iNKT levels isgreater when compared to the elevation resulting from administration ofan equivalent amount of a glycolipid analogue comprising alpha-galactose(αGal) as the glycosyl head group.
 31. A method for stimulatingproduction of one or more cytokines and/or one or more chemokines in ahuman subject in need thereof, the method comprising: administering tothe subject a therapeutically effective amount of a pharmaceuticalcomposition, wherein the composition comprises a compound according toany one of claims 1 to 14 in an amount sufficient to increase cytokineand/or chemokine production.
 32. The method of claim 31, wherein thecytokine production is sufficient to transactivate downstream immunecells.
 33. The method of claim 32, wherein the downstream immune cellscomprise one or more of dendritic cells (DC), natural killer cells (NK),B cells, CD4⁺ T and CD8⁺ T cells.
 34. The method of claim 31, whereinthe cytokines comprise Th1 cytokines.
 35. The method of claim 34,wherein the one or more cytokines are selected from the group consistingof interferon-gamma (IFN-γ), GM-CSF, TNFα, interleukin 2, interleukin 12and interleukin
 10. 36. The method of claim 31, wherein the one or morechemokines are selected from the group consisting of RANTES, MIP-1α, KC,MCP-1, IP-10 and MIG.
 37. The pharmaceutical composition of claim 15,wherein the composition is a vaccine adjuvant.
 38. The pharmaceuticalcomposition of claim 15, wherein the composition is an anti-cancertherapeutic.
 39. The method of claim 16, wherein R⁴ in the compound isselected from substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl, and wherein the compound is capable ofincreasing Th1 cytokines in humans with minimum accompanying increase inTh2 cytokines.
 40. A method for augmenting the immune response by anantigen in a subject, the method comprising administering to the subjectan effective amount of a vaccine comprising one or more antigens and animmunogenic amount of an adjuvant composition of claim
 15. 41. Themethod of claim 40, wherein the one or more antigens are selected fromthe group consisting of bacterial antigen, viral antigen, fungalantigen, protozoal antigen, prion antigen, neoantigen, tumor antigen andself-antigen.
 42. The method of claim 40, wherein the vaccine is in aform selected from the group consisting of a nucleic acid, protein,peptide, glycoprotein, carbohydrate, fusion protein, lipid, glycolipid,carbohydrate-protein conjugate; cells or extracts thereof; dead orattenuated cells, or extracts thereof; tumor cells or extracts thereof;viral particles; and allergens or mixtures thereof.
 43. The method ofclaim 40, wherein the antigen administered is a tumor antigen.
 44. Themethod of claim 40, wherein said amount of vaccine is administered inthe range of 0.1 μg-100 mg per kg of body weight.
 45. The method ofclaim 40, wherein said amount of the adjuvant is in the range of 10-100μg per kg of body weight.
 46. The method of claim 40, wherein thecomposition co-administered is a co-formulated pharmaceuticallyacceptable adjuvant composition comprising the compound having Formula(I) and a pharmaceutically acceptable carrier.
 47. An article ofmanufacture comprising the compound of claim
 1. 48. A kit comprising thecompounds of any one of claims 1-14 and instructions for use.